Delivery of therapeutic compounds to the brain and other tissues

ABSTRACT

The present invention relates to the intrathecal (IT) administration of recombinant enzyme to treat lysosomal storage disorders. In an exemplary embodiment, intrathecal administration of human α-L-iduronidase (rhIDU) injections in MPS I affected animals resulted in significant enzyme uptake, significant rh-iduronidase activity in brain and meninges and a decrease of glycosaminoglycan (GAG) storage in cells of MPS I subjects to that of normal subjects. Intrathecal administration proved more effective than intravenous treatment at alleviating MPS I symptoms, indicating it is a useful method of treating lysosomal storage disorders.

FIELD OF THE INVENTION

The present invention relates to the intrathecal (IT) administration ofrecombinant enzyme to treat lysosomal storage disorders. Furthercontemplated is the induction of antigen specific tolerance prior tointrathecal administration of replacement enzyme.

BACKGROUND OF THE INVENTION

The brain is shielded against potentially harmful substances by theblood-brain barrier (BBB). The microvascular barrier between blood andbrain is made up of a capillary endothelial layer surrounded by abasement membrane and tightly associated accessory cells (pericytes,astrocytes). The brain capillary endothelium is much less permeable tolow-molecular weight solutes than other capillary endothelia due to anapical band of tight association between the membranes of adjoiningcells, referred to as tight junctions. In addition to diminished passivediffusion, brain capillary endothelia also exhibit less fluid-phasepinocytosis than other endothelial cells. Brain capillaries possess fewfenestrae and few endocytic vesicles, compared to the capillaries ofother organs (see Pardridge, J. Neurovirol. 5: 556-569 (1999)). There islittle transit across the BBB of large, hydrophilic molecules aside fromsome specific proteins such as transferrin, lactoferrin and low-densitylipoproteins, which are taken up by receptor-mediated endocytosis (seePardridge, J. Neurovirol. 5: 556-569 (1999)); Tsuji and Tamai, Adv. DrugDeliv. Rev. 36: 277-290 (1999); Kusuhara and Sugiyama, Drug Discov.Today 6:150-156 (2001); Dehouck, et al. J. Cell Biol. 138: 877-889(1997); Fillebeen, et al. J. Biol. Chem. 274: 7011-7017 (1999)).

The blood-brain barrier (BBB) also impedes access of beneficial activeagents (e.g., therapeutic drugs and diagnostic agents) to centralnervous system (CNS) tissues, necessitating the use of carriers fortheir transit. Blood-brain barrier permeability is frequently arate-limiting factor for the penetration of drugs or peptides into theCNS (see Pardridge, J. Neurovirol. 5: 556-569 (1999); Bickel, et al.,Adv. Drug Deliv. Rev. 46: 247-279 (2001)). For example, management ofthe neurological manifestations of lysosomal storage diseases (LSDs) issignificantly impeded by the inability of therapeutic enzymes to gainaccess to brain cell lysosomes. LSDs are characterized by the absence orreduced activity of specific enzymes within cellular lysosomes,resulting in the accumulation of undegraded “storage material” withinthe intracellular lysosome, swelling and malfunction of the lysosomes,and ultimately cellular and tissue damage. Intravenous enzymereplacement therapy (ERT) is beneficial for LSDs (e.g. MPS I, MPS II).However, the BBB blocks the free transfer of many agents from blood tobrain, and LSDs that present with significant neurological sequelae(e.g. MPSI, MPS III, MLD, GM1) are not expected to be as responsive tointravenous ERT. For such diseases, a method of delivering thereplacement enzyme across the BBB and into the lysosomes of the affectedcells would be highly desirable.

There are several ways of circumventing the BBB to enhance braindelivery of an administered active agent include direct intra-cranialinjection, transient permeabilization of the BBB, and modification ofthe active agent to alter tissue distribution. Direct injection of anactive agent into brain tissue bypasses the vasculature completely, butsuffers primarily from the risk of complications (infection, tissuedamage) incurred by intra-cranial injections and poor diffusion of theactive agent from the site of administration. Permeabilization of theBBB entails non-specifically compromising the BBB concomitant withinjection of intravenous active agent and is accomplished throughloosening tight junctions by hyperosmotic shock (e.g. intravenousmannitol). High plasma osmolarity leads to dehydration of the capillaryendothelium with partial collapse of tight junctions, little selectivityin the types of blood-borne substances that gain access to the brainunder these conditions, and damage over the course of a life-longregimen of treatment.

The distribution of an active agent into the brain may also be increasedby transcytosis, the active transport of certain proteins from theluminal space (blood-side) to the abluminal space (brain-side) of theBBB. Transcytosis pathways are distinct from other vesicular trafficwithin the capillary endothelial cell and transit can occur withoutalteration of the transported materials. Transcytosis is a cell-typespecific process mediated by receptors on the BBB endothelial surface.Attachment of an active agent to a transcytosed protein (vector orcarrier) is expected to increase distribution of the active substance tothe brain. In transcytosis, the vector is presumed to have a dominanteffect on the distribution of the joined pair. Vector proteins includeantibodies directed at receptors on the brain capillary endothelium(Pardridge, J. Neurovirol. 5: 556-569 (1999)) and ligands to suchreceptors (Fukuta, et al., 1994, Pharm. Res. 1994; 11(12):1681-8;Broadwell, et al., Exp Neurol. 1996; 142(1):47-65)). Antibody vectorsare transported through the capillary endothelium by a process ofadsorptive endocytosis (non-specific, membrane-phase endocytosis) andare far less efficiently transported than actual receptor ligands, whichcross the BBB by a saturable, energy-dependent mechanism (Broadwell, etal., Exp Neurol. 1996; 142(1):47-65).

Direct administration of proteins into the brain substance has notachieved significant therapeutic effect due to diffusion barriers andthe limited volume of therapeutic that can be administered.Convection-assisted diffusion has been studied via catheters placed inthe brain parenchyma using slow, long-term infusions (Bobo, et al.,Proc. Natl. Acad. Sci. U.S.A 91, 2076-2080 (1994); Nguyen, et al. J.Neurosurg. 98, 584-590 (2003)), but no approved therapies currently usethis approach for long-term therapy. In addition, the placement ofintracerebral catheters is very invasive and less desirable as aclinical alternative.

Intrathecal (IT) injection, or the administration of proteins to thecerebrospinal fluid (CSF), has also been attempted but has yielded onlymoderate success in a few examples of delivery via the CSF [Dittrich etal., Exp. Neurol. 141:225-239 (1996); Ochs et al., Amyotroph. Lateral.Scler. Other Motor Neuron Disord. 1:201-206 (2000); Bowes et al., BrainRes. 883:178-183 (2000)]. For nerve growth factor (NGF), theadministration of the factor into the ventricle of the brain, did havesome beneficial effects on the brain (Koliatsos et al., Exp. Neurol.112, 161-173 (1991), but did not show significant diffusion into thebrain substance. A major challenge in this treatment has been thetendency of the factor to bind the ependymal lining of the ventriclevery tightly which prevented subsequent diffusion. Currently, there areno approved products for the treatment of brain genetic disease bytherapeutic administration directly to the CSF.

The challenges in treating the brain with these and other therapeuticsstudied in the past have suggested that the barrier to diffusion at thebrain's surface, as well as the lack of diffusion and efficacy of braintreatment, were too great an obstacle to achieve adequate therapeuticeffect in the brain for any disease. Prior evidence suggests thatintraventricular or intrathecal enzyme therapy would not worksufficiently to be effective, and in fact, no human studies of thisapproach have been published in the recent past and there are nosuccessful examples of treatment via that route. Intrathecal injectionconfers an advantage over other standard treatment regimens, however, inthat the CSF provides superior access to the brain and meninges. The CSFcovers the brain and provides large surface area contact with corticalneurons up to 6 mm below the surface, allowing for more efficientpenetration of the therapeutic into the brain tissue.

Lysosomal storage disorders affecting the nervous system demonstrateunique challenges in treating these diseases with traditional therapies.There is often a large build-up of glycosaminoglycans (GAGs) in neuronsand meninges of affected individuals, leading to either mild or severeforms of the disease. For example, brain disease in severe MPS Ipatients is characterized by developmental delay, hydrocephalus, severemental retardation, and eventual decline and death due to diseasesymptoms. Mild MPS I brain is characterized by perivascular GAG storage,hydrocephalus, learning disabilities and spinal cord compression due toswelling and scarring from storage disease. In MPS I patients in whichmeningeal storage is affected, the meninges are obstructed, reducing CSFresorption and leading to high pressure hydrocephalus. This aberrantlysosomal storage also leads to thickening and scarring of the meningesfrom storage disease.

In the lysosomal storage disorder, Gaucher disease, patients with thesevere fat of the disease (type 2 and type 3) have brain disease andintravenous enzyme therapy is insufficient to effectively and adequatelytreat the brain. Intrathecal and intraparenchymal enzyme therapy withglucocerebrosidase, the enzyme deficient in Gaucher disease, hassucceeded in getting into the brain but did not successfully treat thebrain storage (Zirzow et al., Neurochem. Res. 24:301-305. 1999). At thistime, no brain disease resulting from a lysosomal disorder hassuccessfully been treated by any means available.

Thus, there remains a need in the art to develop methods whicheffectively treat lysosomal storage disorders through effectiveadministration of enzyme replacement therapy. More particularly, a needexists for more effective methods of administration of compounds andcompositions that can more efficiently deliver active agents to thebrain and central nervous system for the treatment of lysosomal storagedisorders.

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions for thetreatment of central nervous system manifestations of enzyme storagediseases. More particularly, the present invention is based on thediscovery that intrathecal delivery of compositions comprising enzymesthat are deficient or lacking in lysosomal storage disorders, results insustained, long-term clinically useful therapeutic intervention of thecentral nervous system manifestations of such diseases. Thus, thepresent invention is directed to enzyme replacement therapy for suchdiseases by intrathecal administration into the cerebrospinal fluid ofsubjects in need of such therapy.

Accordingly, in one aspect of the present invention, there is provided amethod of treating a lysosomal storage disease comprising providing apharmaceutical composition comprising a protein defective or missing inthe lysosomal storage disease; and delivering the pharmaceuticalcomposition into the cerebrospinal fluid the subject, whereby theprotein is delivered at a level which provides a therapeutic effect inthe mammalian subject. More particularly, the method generally comprisesdelivery of the protein to the brain tissue of the subject at a levelwhich provides a therapeutic effect in the mammalian subject. Morespecifically, the delivery to the cerebrospinal fluid is achieved byintrathecal injection.

In particularly preferred embodiments, the methods of the presentinvention provide for the intrathecal administration of iduronidase toeffect a therapeutic intervention of MPS. This treatment has abeneficial effect on the subject, as it reduces or eliminates glycogenstorage granules in tissues. Moreover, intrathecal injection of theenzyme into the cerebrospinal fluid of both neonatal and adult subjects,results in therapeutic levels of iduronidase in the brain and thereduction or elimination of glycosaminoglycan storage granules in braintissue.

While certain embodiments use iduronidase as the enzyme being replaced,it should be understood that the methods of the present invention may beused for the therapeutic intervention of other diseases that require theadministration of a different enzyme. For example, the present inventionalso contemplates intrathecal administration of beta-glucuronidase (MPSVII), iduronate sulfatase (MPS II), alpha-N-acetylglucosaminidase (MPSIIIB), arylsulfatase A (MLD), glucocerebrosidase, β-glucosidase orN-acetylgalactosamine 4-sulfatase.

Further, it is contemplated that the presence on the cell surface ofbrain cells of a high affinity receptor for the uptake of the enzyme,even at low concentrations of the enzyme will produce a concentrationgradient in the CSF that drives the enzyme to traverse the brain'ssurface across the brain-CSF interface.

In preferred embodiments, the present invention is directed to a methodfor treating a lysosomal storage disease in a mammal comprisingintrathecal administration into the central nervous system of the mammala pharmaceutical composition comprising an enzyme that is deficient inthe lysosomal storage disease in an amount effective to ameliorate thesymptoms of the lysosomal storage disease. Those of skill in the artroutinely monitor subjects for symptoms of lysosomal storage diseasethrough routine assessment of history, physical examination,echocardiography, electrocardiography, magnetic resonance imaging,polysomnography, skeletal survey, range of motion measurements, cornealphotographs, and skin biopsy (see U.S. Pat. No. 6,585,971). Any suchmethods may be used in conjunction with the treatment methods of thepresent invention.

Preferably, the enzyme replacement therapy pharmaceutical composition isadministered in an amount effective to decrease the amount of storagegranules present in the brain tissue of the mammal. More particularly,the therapy results in a reduction of GAG build-up in the neuronaland/or meningeal tissue of the subject. In certain preferred methods ofthe invention, the therapeutic intervention ameliorates high pressurehydrocephalus associated with lysosomal storage disease. Preferably, theintrathecal administration of the enzyme therapy of the presentinvention produces a reduction in meningeal swelling that results fromthe presence of lysosomal storage granules in the meninges ofindividuals suffering from lysosomal storage disease.

The methods of the present invention may be used for the treatment ofany lysosomal storage disease which manifests an effect in brain ormeningeal tissue and requires the medicament to enter the brain ormeninges. The methods of the present application, achieve a therapeuticeffect by crossing the brain-CSF interface and ameliorating thedeleterious effects of the lysosomal storage disease in brain tissue.For example, such a disease may include, but is not limited toaspartylglucosaminuria, cholesterol ester storage disease, Wolmandisease, cystinosis, metachromatic leukodystrophy, Danon disease, Fabrydisease, Farber lipogranulomatosis, Farber disease, fucosidosis,galactosialidosis types I/II, Gaucher disease types I/II/III, Gaucherdisease, globoid cell leukodystrophy, Krabbe disease, glycogen storagedisease II, Pompe disease, GM1-gangliosidosis types I/II/III,GM2-gangliosidosis type I, Tay Sachs disease, GM2-gangliosidosis typeII, Sandhoff disease, GM2-gangliosidosis, α-mannosidosis types I/II,β-mannosidosis, metachromatic leukodystrophy, mucolipidosis type I,sialidosis types I/II mucolipidosis types II/III I-cell disease,mucolipidosis type IIIC pseudo-Hurler polydystrophy,mucopolysaccharidosis type I, mucopolysaccharidosis type II, Huntersyndrome, mucopolysaccharidosis type IIIA, Sanfilippo syndrome,mucopolysaccharidosis type IIIB, mucopolysaccharidosis type IIIC,mucopolysaccharidosis type IIID, mucopolysaccharidosis type IVA, Morquiosyndrome, mucopolysaccharidosis type IVB Morquio syndrome,mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, Slysyndrome, mucopolysaccharidosis type IX, multiple sulfatase deficiency,neuronal ceroid lipofuscinosis, CLN1 Batten disease, Niemann-Pickdisease types A/B, Niemann-Pick disease, Niemann-Pick disease type C1,Niemann-Pick disease type C2, pycnodysostosis, Schindler disease typesI/II, Schindler disease, and sialic acid storage disease.

In particularly preferred embodiments, the disease ismucopolysaccharidosis, and more preferably, the disease ismucopolysaccharidosis I. In certain embodiments, the subject with thelysosomal storage disease has a diminished normal α-L-iduronidaseactivity. The activity may be diminished because the enzyme is mutatedor absent in the subject. In particular embodiment, the mammal has about50% or less of a normal α-L-iduronidase activity. In other embodiments,the subject has 75% or less of a normal α-L-iduronidase activity. Inorder to treat this deficiency, the methods of the present invention mayemploy a pharmaceutical composition which comprises a dose of at leastabout 125,000 units or 0.5 mg/kg of the human α-L-iduronidase. Otherpreferred doses include between about 0.01 mg/15-20 kg body weight ofthe subject to about 10 mg/15-20 kg body weight of the subject. The dosemay be administered in any convenient dose and at any convenientlyspaced interval determined by the physician administering the treatment.In certain embodiments, the enzyme replacement therapy is administeredweekly to a subject suffering from a deficiency in a lysosomal storageenzyme.

In certain exemplary embodiments, the pharmaceutical compositioncomprises a dose of at least about dose of between about 0.01 mg/15 ccof CSF to about 5.0 mg/15 cc of CSF in the mammal of the humanα-L-iduronidase is administered weekly to a subject suffering from adeficiency thereof. Preferably, the pharmaceutical composition comprisesa dose of about 1 mg/15 cc of CSF in the mammal of the humanα-L-iduronidase is administered weekly to a subject suffering from adeficiency thereof. An exemplary pharmaceutical composition isformulated in a buffer comprising 0.58 mg/ml iduronidase in a buffercomprising 100 mM sodium phosphate, 150 mM NaCl and 0.001% polysorbate80.

The pharmaceutical compositions for use in the methods of the presentinvention also may contain other components, such as for example, humanalbumin. In particular embodiments, the compositions contain humanalbumin at a concentration of at least about 1 mg/mL. the compositionsmay be in the form of buffered solutions, such as for example, in abuffered solution comprising a sodium phosphate buffer at aconcentration of about 10-50 mM.

In specific embodiments, the lysosomal storage disorder is MPS 1 and theenzyme is recombinant iduronidase administered intrathecally in anamount of about 0.5 μg to about 20 mg per kilogram of body weight. Inspecific embodiments, the amount is about 0.5 μg to about 0.5 mg perkilogram of body weight. More particularly, it is contemplated that therecombinant iduronidase is administered in a dosage of about 1.0 μg to100 μg, 2.0 μg to 50 μg, or 10 μg to 100 μg per kilogram of body weight.These are merely exemplary amounts of iduronidase and those of skill inthe art will understand that these doses may be varied depending on ageof the subject, size of the subject, stage of the disease and the like.In preferred embodiments, the recombinant iduronidase is administered ina dosage of about 1.0 μg to 15 mg, 2.0 μg to 10 mg, or 10 μg to 5 mg.

The enzyme for the replacement therapy may be prepared from any sourcecommonly used for the preparation of such enzymes. In certainembodiments, the enzyme is iduronidase that is secreted and purifiedfrom mammalian cells in culture transfected with a DNA sequence encodinghuman iduronidase.

The enzyme delivered in the intrathecal methods of treatment of thepresent invention may be administered through any convenient routecommonly used for intrathecal administration. For example, theintrathecal administration may be via a slow infusion of at least 0.5mg/kg of the formulation for about an hour. However, it should beunderstood that the dosage may vary from about 0.01 mg/15-20 kg bodyweight of the subject to about 10 mg/15-20 kg body weight of the subjectover similar infusion rates. Advantageously, administering theintrathecal enzyme replacement therapy results in the normalization oflysosomal storage granules in the neuronal and/or meningeal tissue ofthe subjects as discussed above. In particularly preferred embodiments,it is contemplated that the deposition of storage granules isameliorated from neuronal and glial tissue, thereby alleviating thedevelopmental delay and regression seen in individuals suffering withlysosomal storage disease. Other preferred embodiments results in thenormalization of lysosomal storage granules in the cerebral meningesnear the arachnoid granulation, the presence of which in lysosomalstorage disease result in high pressure hydrocephalus. Therefore, themethods of the invention are directed to the treatment of such highpressure hydrocephalus associated with lysosomal storage disease. Themethods of the invention also may be used in treating spinal cordcompression that results from the presence of lysosomal storage granulesin the cervical meninges near the cord at C1-C5 or elsewhere in thespinal cord. The methods of the invention also are directed to thetreatment of cysts that are caused by the perivascular storage oflysosomal storage granules around the vessels of the brain.

In other embodiments, the therapy also may advantageously result innormalization of liver volume and urinary glycosaminoglycan excretion,reduction in spleen size and apnea/hypopnea events, increase in heightand growth velocity in prepubertal subjects, increase in shoulderflexion and elbow and knee extension, and reduction in tricuspidregurgitation or pulmonic regurgitation. For methods of monitoring sucheffects, those of skill in the art are specifically referred to Example5 of U.S. Pat. No. 6,585,971, which is incorporated herein by referencespecifically for teachings of Example 5, and more generally for teachingmethods and compositions of formulating recombinant iduronidase.

In preferred embodiments, the therapeutic administering in the presentapplication involves administration of human recombinantα-L-iduronidase, which reduces lysosomal storage in at least the braintissue of the individual having the lysosomal storage disease. In thosepreferred aspects of the invention in which iduronidase is beingadministered intrathecally into the CSF, the composition being deliveredcomprises about 1 mg iduronidase/20 kg of body weight of the mammalbeing treated for MPS. In particular embodiments, the above dose isdelivered to 15 cc CSF. At such a concentration it is contemplated thatthe enzyme concentration will be 18,000 units per ml of CSF. It shouldbe understood that the aforementioned dosage is merely an exemplarydosage and those of skill in the art will understand that this dosagemay be varied.

The intrathecal administration of the present invention may compriseintroducing the pharmaceutical composition into a cerebral ventricle.Alternatively, the intrathecal administration may comprise introducingthe pharmaceutical composition into the lumbar area. In yet anotheralternative, the intrathecal administration comprises introducing thepharmaceutical composition into the cisterna magna. Any suchadministration is preferably via a bolus injection. Depending on theseverity of the symptoms and the responsiveness of the subject to thetherapy, the such a bolus injection may be administered once per week,once per month, once every two months, once every three months, onceevery 6 months or annually. In other embodiments, the intrathecaladministration is achieved by use of an infusion pump. Thepharmaceutical could of course be intrathecally administered continuallyover a period of at least several days or alternatively, the intrathecaladministration is continually over a period of at least four weeks. Ofcourse, where the administration is via continuous infusion, the rate ofdose administration of the enzyme replacement therapy may be greatlyreduced as compared to the bolus injection administration.

In certain embodiments, the therapeutic regimens may be such that theintrathecal administration is combined with systemic administration ofthe pharmaceutical composition comprising said enzyme that is deficientin the disease in combination. In preferred such embodiments, theintrathecal administration may be performed on a monthly basis althoughother time intervals between administration also are contemplated.Preferably, the systemic administration in such combined administrationregimens is intravenous administration. In specific embodiments, themethods of the invention contemplate treatment of a lysosomal storagedisease by administering an enzyme such as rh-IDU intrathecally toeffect delivery into the CNS and systemically to ameliorate the effectsof the lysosomal storage disease in non-CNS sites. For example, inspecific embodiments, the rh-IDU is administered intrathecally on amonthly basis and intravenously on a fortnightly, weekly, daily, orevery other day basis. In certain embodiments, the subject may betolerized to the intrathecal and/or rh-IDU administration using animmunosuppressive tolerization regimen prior to initiation of thetherapeutic regimen.

The methods of the present invention are preferably for the therapeuticintervention of a human suffering for a lysosomal storage disease.

In preferred embodiments of the invention, the enzyme being delivered tothe cerebrospinal fluid naturally comprises or has been engineered tocomprise a component that allows the uptake of the enzyme by a highaffinity receptor. For example, the enzyme comprises or has beenengineered to comprise a moiety that allows said enzyme to bind to areceptor selected from a mannose-6-phosphate receptor, melanotransferrinreceptor, and LRP receptor or any other receptor that is ubiquitouslyexpressed on the surface of brain cells. In preferred embodiments, theenzyme comprises mannose-6-phosphate moieties that allow the enzyme tobe taken up by a cell that expresses a mannose-6-phosphate receptor. Inan alternative embodiment, the enzyme naturally binds, or has beenengineered to possess GAG binding capacity. In an alternativeembodiment, the enzyme comprises p97, RAP, transferrin or IGF2.

In certain aspects of the invention, the subjects being treated withenzyme replacement therapy for lysosome storage disease are renderedtolerant to such therapy using tolerization regimens.

In certain embodiments of the invention, the enzyme used for the enzymereplacement therapy in the lysosomal storage disease is one whichnaturally comprises, or is fused to, a moiety that facilitates the highuptake of the enzyme. In preferred embodiments, such an enzyme isiduronidase, whether recombinant or wild-type. The moiety thatfacilitates uptake of the enzyme may be any moiety such as a bindingpartner of a ligand or receptor expressed on the surface of the cell tobe targeted for the therapy. In particularly preferred embodiments, themoiety is selected from the group consisting is a mannose-6-phosphateresidue, a RAP polypeptide, and a p97 polypeptide. Other aspects of thepresent invention define methods which further comprise inducing antigenspecific tolerance prior to the enzyme replacement therapy. Suchinduction of tolerance to the therapy may employ administration of animmunosuppressive agent, such as e.g., cyclosporine either alone or incombination with an agent such as azathioprine, which may haveantiproliferative and/or co-stimulatory signal blocking effects.

Specific embodiments of the present invention contemplate methods ofpromoting the breakdown of glycosaminoglycan (GAG) in a brain cell of asubject having lysosomal storage disease, the method comprisingintrathecally administering to the subject a pharmaceutical compositioncomprising an enzyme deficient in the lysosomal storage disease in anamount effective to reduce the amount of GAG present in the brain cellas compared to the amount of GAG present in the cell prior to theadministration.

In specific embodiments throughout the present specification it shouldbe understood that the methods of the invention may be used to reduceGAG storage and/or promote GAG breakdown in any brain cell that has anabnormal storage of GAG. The brain cells may be neuron, neuroglia, orependymal cells. In specific embodiments, the brain cell may be selectedfrom at least one of the group consisting of neurons, glial cells,microglial cells, astrocytes, oligodendroglial cells, perivascularcells, perithelial cells, meningeal cells, ependymal cells, arachnoidgranulation cells, arachnoid membranes, dura mater, pia mater andchoroid plexus cells. In preferred embodiments, the brain cell is ameningeal cell. It is contemplated that in certain embodiments, thesubject has high pressure hydrocephalus and the administering reducesthe amount of CSF fluid in the meningeal tissue of the subject. In otherembodiments, it is contemplated that the subject has spinal cordcompression and the administering reduces or otherwise alleviates thesymptoms of said compression. In preferred embodiments, the therapeuticmethods of the invention reduce the number of lysosomal storage granulesin the cell as compared to the number of lysosomal storage granulespresent in a similar cell in the absence of the intrathecaladministration.

Another embodiment of the invention contemplates a method of decreasingmeningeal swelling in a subject having a lysosomal storage disease themethod comprising intrathecally administering to the subject apharmaceutical composition comprising an enzyme deficient in thelysosomal storage disease in an amount effective to decrease meningealinflammation of the subject as compared to the size of the meninges ofthe subject prior to the administration. The subject may be a humansubject.

Other beneficial aspects of the invention contemplate methods ofdecreasing spinal cord compression in a subject suffering from alysosomal storage disease the method comprising intrathecallyadministering to the subject a pharmaceutical composition comprising anenzyme deficient in the lysosomal storage disease in an amount effectiveto decrease meningeal inflammation of the subject as compared to thesize of the meninges of the subject prior to the administration. Inthese and other methods of the invention, the motor skills of thesubject are preferably improved with the administration of thepharmaceutical composition as compared to the motor skills of the animalprior to the administration of the pharmaceutical composition.

The foregoing paragraphs are not intended to define every aspect of theinvention, and additional aspects are described in other sections, suchas the Detailed description.

In addition to the foregoing, the invention includes, as an additionalaspect, all embodiments of the invention narrower in scope in any waythan the variations defined by specific paragraphs above. For example,certain aspects of the invention that are described as a genus, and itshould be understood that every member of a genus is, individually, anaspect of the invention. Although the applicants invented the full scopeof the invention described herein, the applicants do not intend to claimsubject matter described in the prior art work of others. Therefore, inthe event that statutory prior art within the scope of a claim isbrought to the attention of the applicants by a Patent Office or otherentity or individual, the applicants reserve the right to exerciseamendment rights under applicable patent laws to redefine the subjectmatter of such a claim to specifically exclude such statutory prior artor obvious variations of statutory prior art from the scope of such aclaim. Variations of the invention defined by such amended claims alsoare intended as aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further illustrate aspects of the present invention. Theinvention may be better understood by reference to the drawings incombination with the detailed description of the specific embodimentspresented herein.

FIG. 1 describes enzyme levels in brain of canine subjects afterintrathecal injection.

FIG. 2 describes levels of rh-iduronidase measured in deep brain andsurface brain tissues in canines.

FIG. 3 describes levels of iduronidase activity in spinal cord andspinal meninges of iduronidase-treated MPS I animals.

FIG. 4 describes a comparison of glycosaminoglycan (GAG) levels in MPS Itreated animals receiving either intrathecal or IV administration ofiduronidase.

FIG. 5 depicts electron microscopy (in brain sections) of GAG storage inperivascular macrophage disease of iduronidase treated and untreated MPSI animals.

FIG. 6 is a greater magnification of electron microscopy (brainsections) of GAG storage in perivascular macrophage disease ofiduronidase treated and untreated MPS I animals, which demonstrates thatperivascular cells in treated MPS I animals lack GAG storage.

FIG. 7 is a comparison of neuron disease pathology in iduronidasetreated and untreated MPS I animals which shows that treated animals arefree of lamellar GAG storage.

FIG. 8 is a comparison of brain sections assessed for meningeal diseasein treated or untreated MPS I animals which shows the absence of largeGAG-filled foam cells in the meninges of treated animals, as compared tocontrols.

FIG. 9 illustrates that brain sections of MPS I treated animals exhibitminor lymphocytic infiltrate into the meninges.

FIG. 10A through 10C provide comparisons of the effects of monthly vs.weekly intrathecal administration of rhIDU. FIG. 10A shows GAG levels inbrain were reduced to normal with treatment (*p=0.003). FIG. 10B showsGAG levels in spinal cord were reduced with treatment (p=0.22). FIG. 10Cshows GAG levels in spinal meninges were reduced with treatment(*p=0.02).

FIGS. 11A and 11B show comparison of GAG between untreated andintrathecally treated dogs. GAG storage is visibly reduced inperivascular cells, glia, and neocortical leptomeninges in treated dogs.The untreated samples (FIG. 11A) show foamy, swollen, GAG laden cells ascontrasted with the treated samples shown in FIG. 11B which are thincells with markedly less storage.

FIG. 12A through 12D shows that immune tolerance reduces theinflammatory response to intrathecally administered rhIDU. A lymphocyticand plasmocytic infiltrate develops in treated dogs (FIGS. 12A and 12C).Pre-conditioning with a regimen to induce immune tolerance greatlyreduces this response (FIGS. 12B and 12D).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for treating lysosomal storagedisorders using intrathecal injection of enzymes deficient in theparticular disorder being treated. The method can be coupled with atolerance inducing regimen to provide a more effective treatment tosubjects.

The present application is based on the discovery that intrathecaldelivery of enzyme replacement therapy for lysosomal storage disordersresults in sustained, long-term clinically useful therapeuticintervention of the central nervous system manifestations of suchdiseases. Properties of the enzyme such as solubility and binding tosubstrates can have an impact on the penetration of the enzyme intobrain tissue and traversal of the therapeutic agent across the CSF-braininterface.

Thus, for the first time, the present specification details thesuccessful treatment of the lysosomal storage diseases within the brainand meninges using recombinant iduronidase, thereby showing for thefirst time that it is possible to treat the brain and meningeal diseasein storage diseases such as MPS I. Given that peripheral enzyme therapyfor MPS I was approved for human use in 2003, the present inventionallows the immediate translation to the treatment of human MPS Ipatients in clinical trials using the same recombinant iduronidasecompositions presently approved for peripheral therapy. This representsan important advance in the treatment of lysosomal storage diseasessince brain manifestations of lysosomal storage diseases have, to date,been refractory to treatment. While many of the exemplary methodsdescribed herein are exemplified using studies performed on MPS I, it iscontemplated that the present findings may be extended to the treatmentof other lysosomal storage disorders for which enzyme therapies arecurrently in development.

In particular embodiments, the instant specification details for thefirst time that the enzyme iduronidase can penetrate the brain betterthan other enzymes that have previously been attempted. In particularpreferred embodiments, iduronidase is used to treat MPS disorders.Intrathecal administration of iduronidase is contemplated due to theability of this enzyme to bind GAG in brain tissue, which may provide abinding site to pull the enzyme into the tissue fluid space. Thepresence of mannose-6-phosphate moieties on the iduronidase allows ahigh affinity uptake of the enzyme from the CSF, thereby allowing smallconcentrations of the enzyme in the CSF to have a clinically therapeuticeffect. The fact that mannose-6-phosphate moieties bind to a highaffinity receptor on the surface of nearly all cells allows even smallamounts of iduronidase to be taken up by brain and meninges and becorrective of lysosomal storage disease at those sites.

Iduronidase demonstrates an extremely high affinity for its receptorwith half maximal binding at concentration of approximately 1 nanomolar(12 units/mil) and moreover, given the half maximal correction of thedefect at approximately 1 picomolar, the addition of even small amountsof enzyme into the CSF space would create an enormous gradient drivingenzyme into the brain. In an exemplary treatment regimen, at a 1 mg dosein a 20 kg dog with 15 cc of CSF, the enzyme concentration in the CSF ispredicted to be about 18,000 units/ml. This concentration is more than1,000 fold above the concentration needed to observe uptake and1,000,000 fold above the concentration required for half maximalcorrection. Therefore, even an inefficient process in which only 1% ofthe enzyme penetrates the brain would result in levels in the brain thatare 10 times the uptake constant, a concentration that should driveefficient uptake and about 10,000 fold above the half maximal correctionconcentration. Given this easily achievable gradient, the effects of theproperties of the enzyme on diffusion, and the low concentration neededfor uptake and correction, it is demonstrated herein that iduronidasesuccessfully treats symptoms of MPS I in vivo. One additional advantagein using intrathecal iduronidase therapy in treating MPS I is that MPS Idisease enhances the permeability of the brain surface to enzyme therapywhich makes intrathecal therapy an attractive method of treating MPS I.

The significance of the large concentration gradient resulting fromintrathecal delivery of enzyme combined with the small but significantpenetration of the enzyme into the brain could be sufficient to achievetherapeutic efficacy in any lysosomal storage disorder. The effect ofthis large concentration gradient generated by the high-uptake receptorbinding property of iduronidase had not been appreciated prior to thepresent invention and is important in understanding how the brain can beforce-fed enzyme across the ependymal layer and how a large number ofenzymes may now be driven to diffuse across the blood brain barrier.Methods and compositions for achieving such correction with iduronidase,as well as other enzymes for lysosomal storage diseases are discussed infurther detail herein below.

DEFINITIONS

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods described, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting, since the scope of the presentinvention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, subject to any specifically excluded limit in the statedrange.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “a,”, and “the” include plural referents unless thecontext clearly dictates otherwise.

By “lysosomal storage disease” is meant any disease resulting from thedeficiency of one or more lysosomal enzymes necessary for metabolizingnatural macromolecules. These diseases typically result in theaccumulation of un-degraded molecules in the lysosomes, resulting inincreased numbers of storage granules (also termed storage vesicles).These diseases are described in more detail below.

A “subject” is meant to include any animal that is to be treated usingthe methods of the invention. Preferably, the subject is a mammaliansubject, including, without limitation, humans and nonhuman primatessuch as chimpanzees and other apes and monkey species; farm animals suchas cattle, sheep, pigs, goats and horses; domestic mammals such as dogsand cats; laboratory animals including rodents such as mice, rats andguinea pigs, and the like. The term does not denote a particular age orsex. Thus, adult and newborn subjects, as well as fetuses, whether maleor female, are included within the term “subject.”

By “therapeutically effective,” the present specification intends todenote any therapeutic benefit that arises as a result of the treatmentmethods of the present invention. For example, such an effect can be thebeneficial effects that manifest in an appropriate target tissue, of theenzyme which is deficient or missing in the lysosomal disorder ofinterest, where such beneficial physiological effect is compared to thatphysiological parameter being measured in the absence of the enzymereplacement therapy. Such a therapeutic effect may be any reduction orelimination of one or more clinical or subclinical manifestations of thedisease of interest. For example, a reduction in the number of storagevesicles (also termed storage granules), or elimination thereof, willprovide a therapeutic benefit to the treated subject. Methods fordetecting the presence of storage granules in a tissue of interest arewell known in the art and described further below in the examples. Suchmethods entail microscopic examination of tissue sections. See, e.g.,Vogler et al. (1990) Am J Pathol 136: 207-217. Moreover, reduction inthe accumulation of substances due to the particular enzyme deficiencyin question, will also confer a therapeutic benefit in the treatedsubject. Such substances may be readily detected using known assays. Forexample, MPS VII results in a build-up of un-degraded glycosaminoglycans(GAGs). GAG levels can be readily measured using methods developed byFarndale et al. (Farndale et al. (1982) Con Tissue Res 9: 247-248) andPoorthuis et al. (Poorthuis et al. (1994) Pediatr Res 36: 187-193).

METHODS OF THE INVENTION

The present invention is directed to novel methods for the treatment oflysosomal storage diseases by providing intrathecal administration ofenzymes defective or missing in such lysosomal storage disorders,thereby providing for the replacement of the defective or missing enzymein the brain tissues of the subject being treated. Delivery to the braintarget tissues is through an intrathecal route of administration. Thesemethods effectively provide for the elimination or reduction of storagegranules in the brain tissues of the treated subject.

Lysosomal storage diseases that may be treated using the methods of theinvention include, but are not limited to, Gaucher's disease (see, e.g.,Barranger et al. Neurochemical Res. (1999) 24:601-615 and NTH TechnologyAssessment Conference Statement, Feb. 27, 1995-Mar. 1, 1995) includingTypes 1, 2 and 3, Fabry's disease (see, e.g., Takanaka et al. Exp. Hem.(1999) 27:1149-1159, Ziegler et. al. Hum. Gene Ther. (1999) 10:1667-1682and Takanaka et al. Hum. Gene Ther. (1999) 10:1931-1939), Tay-Sachsdisease (see, e.g., Guidotti et al. Hum. Mol. Gen. (1999) 8:831-838 andDaly et al. Proc. Natl. Acad. Sci USA (1999) 96:2296-2300), Neimann-PickDisease, Types A, B and C, ornithine-δ-aminotransferase (OAT) deficiency(see, e.g., Jensen et al. Hum. Gene Ther. (1997) 8:2125-2132, Locrazzaet al. Gene Ther. (1995) 2:22-28, Rivero et al. Hum. Gene Ther. (1994)5:701-707), hereditary homocysteinemia (see, e.g., McGill et al. Am. J.Med. Gen. (1990) 36:45-52, Eikelboom et al. Ann. Int. Med. (1999)131:363-365, Watanabe et al. Proc. Nat'l Acad. Sci. USA (1995)92:1585-1589), Mannosidoses, Fucosidoses, Sialodosis, the Mucolipidoses,such as I-cell Disease (Mucolipidoses II) and Pseudo-HurlerPolydystrophy (Mucolipidoses III), Acid Lipase Deficiency, such asWolman Disease and Cholesterol Ester Storage Disease, SulfatideLipidosis including Metachromatic Dystrophy and Multiple SulfataseDeficiency, MPS I (Hurler's disease) (see e.g., Lutzko et al. Hum. GeneTher. (1999) 10:1521-1532, Hartung et al. Hum. Gene Ther. (1999)10:2163-2172), MPS II (Hunter syndrome) (see e.g., Rathmann et. al. Am.J. Hum. Genet. (1996) 59:1202-1209, Stronicek et al. Transfusion (1999)39:343-350, Li et al. J. Med. Genet. (1999) 36:21-27), MPS III(Sanfilippo syndrome) (see e.g., Scott et al. Nat. Genet. (1995)11:465-467, Jone et al. J. Neuropath. Exp. Neur. (1997)56(10):1158-1167), MPS IV (Morquoi's syndrome) (see e.g., Nothover etal. J. Inherit. Metab. Dis. (1996) 19:357-365), MPS V (Scheie'ssyndrome) (see e.g., Dekaban et al. Arch. Pathol. Lab. Med. (1976)100:231-245), MPS VI (Maroteaux-Lamy syndrome) (see e.g., Hershovitz etal. J. Inherit. Metab. Dis. (1999) 22:50-62, Villani et al. Biochim.Biophys. Acta. (1999) 1453:185-192, Yogalingam et al. Biochim. Biophys.Acta. (1999) 1453:284-296), and MPS VII (Sly syndrome) (see, e.g. Watsonet al. Gene Ther. (1998) 5:1642-1649, Elliger et al. Gene Ther. (1999)6:1175-1178, Stein et al. J. Virol. (1999) 73 (4):3424-3429, Daly et al.PNAS (1999) 96:2296-2300, Daly et al. Hum. Gene Ther. (1999) 10:85-94);and Sandhoff disease.

A detailed review of the genetic etiology, clinical manifestations, andmolecular biology of the lysosomal storage diseases are detailed inScriver et al., eds., The Metabolic and Molecular Basis of InheritedDisease, 7^(th) Ed., Vol. II, McGraw Hill, (1995). Thus, the enzymesdeficient in the above diseases are known to those of skill in the art,some of these are exemplified in the Table below:

Lysosomal Storage Disease Protein deficiency Mucopolysaccharidosis typeI L-Iduronidase Mucopolysaccharidosis type II Hunter syndromeIduronate-2-sulfatase Mucopolysaccharidosis type IIIA Sanfilipposyndrome Heparan-N-sulfatase Mucopolysaccharidosis type IIIB Sanfilipposyndrome α-N-Acetylglucosaminidase Mucopolysaccharidosis type IIICSanfilippo syndrome AcetylCoA: N-acetyltransferase Mucopolysaccharidosistype IIID Sanfilippo syndrome N-Acetylglucosamine 6-sulfataseMucopolysaccharidosis type IVA Morquio syndrome Galactose 6-sulfataseMucopolysaccharidosis type IVB Morquio syndrome β-GalactosidaseMucopolysaccharidosis type VI N-Acetylgalactosamine 4-sulfataseMucopolysaccharidosis type VII Sly syndrome β-GlucuronidaseMucopolysaccharidosis type IX hyaluronoglucosaminidaseAspartylglucosaminuria Aspartylglucosaminidase Cholesterol ester storagedisease/Wolman disease Acid lipase Cystinosis Cystine transporter Danondisease Lamp-2 Fabry disease α-Galactosidase A FarberLipogranulomatosis/Farber disease Acid ceramidase Fucosidosisα-L-Fucosidase Galactosialidosis types I/II Protective protein Gaucherdisease types I/IIIII Gaucher disease Glucocerebrosidase (β-glucosidase)Globoid cell leukodystrophy/Krabbe disease Galactocerebrosidase Glycogenstorage disease II/Pompe disease α-Glucosidase GM1-Gangliosidosis typesI/II/III β-Galactosidase GM2-Gangliosidosis type I/Tay Sachs diseaseβ-Hexosaminidase A GM2-Gangliosidosis type II Sandhoff diseaseβ-Hexosaminidase A GM2-Gangliosidosis GM2-activator deficiencyα-Mannosidosis types I/II α-D-Mannosidase β-Mannosidosis β-D-MannosidaseMetachromatic leukodystrophy Arylsulfatase A Metachromaticleukodystrophy Saposin B Mucolipidosis type I/Sialidosis types I/IINeuraminidase Mucolipidosis types II/III I-cell diseasePhosphotransferase Mucolipidosis type IIIC pseudo-Hurler polydystrophyPhosphotransferase γ-subunit Multiple sulfatase deficiency Multiplesulfatases Neuronal Ceroid Lipofuscinosis, CLN1 Batten disease Palmitoylprotein thioesterase Neuronal Ceroid Lipofuscinosis, CLN2 Batten diseaseTripeptidyl peptidase I Niemann-Pick disease types A/B Niemann-Pickdisease Acid sphingomyelinase Niemann-Pick disease type C1 Niemann-Pickdisease Cholesterol trafficking Niemann-Pick disease type C2Niemann-Pick disease Cholesterol trafficking Pycnodysostosis Cathepsin KSchindler disease types I/II Schindler disease α-Galactosidase B Sialicacid storage disease sialic acid transporter

Thus, the lysosomal storage diseases that can be treated or preventedusing the methods of the present invention include, but are not limitedto, Mucopolysaccharidosis I (MPS I), MPS II, MPS IIIA, MPS IIIB,Metachromatic Leukodystrophy (MLD), Krabbe, Pompe, CeroidLipofuscinosis, Tay-Sachs, Niemann-Pick A and B, and other lysosomaldiseases as listed above. In particularly preferred embodiments, theenzyme is a lysosomal storage enzyme, such as α-L-iduronidase,iduronate-2-sulfatase, heparan N-sulfatase, α-N-acetylglucosaminidase,arylsulfatase A, galactosylceramidase, acid-alpha-glucosidase,tripeptidyl peptidase, hexosaminidase alpha, acid sphingomyelinase,α-galactosidase, or any other lysosomal storage enzyme.

In even more preferred embodiments, the disease to be treated is MPS Iand the enzyme being replaced is iduronidase. Those of skill in the artare aware of compositions of comprising iduronidase, see for example,U.S. Pat. No. 6,585,971; U.S. Pat. No. 6,569,661; U.S. Pat. No.6,524,835; U.S. Pat. Nos. 6,426,208; 6,238,662; U.S. Pat. No. 6,149,909.Each of the aforementioned patents is incorporated herein by referenceas providing teachings of the iduronidase compositions that may be usedin the methods of the invention. Iduronidase also is availablecommercially as ALDURAZYME™. The iduronidase may be naturally occurringiduronidase that has been isolated from an animal source oralternatively, may be recombinantly produced iduronidase, as producedaccording to exemplary methods described in the above-referencedpatents. In certain embodiments, the iduronidase may be producedrecombinantly in mammalian cells (e.g., as described in the abovepatents) or plant cells (e.g., as described in U.S. Pat. No. 5,929,304.)

In preferred embodiments, the methods of the invention reduce lysosomalstorage granules in the meningeal and/or neuronal tissue of anindividual manifesting lysosomal storage disease. In one sense,therefore, the invention comprises methods of reducing the size ofmeningeal and/or neuronal tissue of a subject having lysosomal storagedisease, the method comprising intrathecally administering to thesubject a pharmaceutical composition comprising an enzyme that isdeficient in the lysosomal storage disease. In other embodiments, theinvention also is directed to reducing lysosomal storagedisease-associated high pressure hydrocephalus in a subject by providingto the subject an intrathecal administration of a pharmaceuticalcomposition comprising an enzyme deficient in the lysosomal storagedisease. Preferably, the enzyme is iduronidase. A therapeuticallyeffective amount of iduronidase in these contexts is any amount ofiduronidase that produces a detectable decrease in lysosomal storagegranules, decreases meningeal and/or neuronal mass, reduces swellingassociated with CSF present in the meninges of individual suffering fromlysosomal disorder associated hydrocephalus and the like. Methods ofdetermining whether the meninges of a subject are swollen are well knownto those of skill in the art, and may include, for example, CAT scans.

In particular embodiments, the intrathecal administration discussedherein is used for the treatment of symptoms that result from lysosomalstorage granules in neuronal, glial or other brain tissues of an animal.Such storage granules manifests in developmental delay and/or regressionin development of the subject suffering from the disease. These symptomsand alleviation thereof with the treatment methods contemplated hereinmay be clinically assessed, for example using Bayley's Scales of InfantDevelopment II, which includes monitoring a motor and developmentalquotient. Development also may be assessed by monitoring language orother intellectual and motor developments. Evoked potential tests suchas auditory or other evoked potential testing also may be used to assessthe effects of the therapy on developmental delay and/or regression.

Other embodiments of the invention contemplate treatment of highpressure hydrocephalus caused by the presence of storage granules in thecerebral meninges near the arachnoid granulations. Such treatment may bemonitored and assessed using art-recognized methods for determining CSFpressure via lumbar puncture and/or via an intraventricular catheter.Any release or reduction in the CSF pressure as a result of thetherapeutic regimens of the present invention will be considered to be atherapeutic benefit of the present invention.

Treatment methods of the invention also are directed to ameliorating theeffects of lysosomal storage in the cervical meninges near the cord atC1-C5 or elsewhere along the cord. Such storage results in symptomsassociated with high pressure of CSF and also other symptoms associatedwith spinal cord compression. The storage results in progressivecompressive spinal cord compression with lower extremity weakness, lossof bowel and bladder control and sensory deficits. Such symptoms may bemonitored using e.g., neurological examination for abnormal Babinski'sreflexes, deep tendon reflexes, motor function or sensation.Neurophysiological deficits of spinal cord compression may be assessedusing somatosensory evoked potentials. Alternatively, magnetic resonanceimaging with or without a contrast agent may be used to identify theanatomic location of compression as well as an evaluation of edema orother indicia of cord injury at the site of compression. The highpressure exerted by the CSF will lead to physiological manifestationssuch as headache, edema and the like. Any reduction in the pressureexerted by CSF, reduction in edema, or any improvement in theneurophysiological deficits, tendon reflexes, motor function orsensation observed as a result of the administration of the therapeuticregimen will be considered to be a therapeutically beneficial effect ofthe methods of the present invention. The subject may particularly bemonitored for any level of improvement in lower extremity weakness,bowel and bladder control and sensory deficits associated with spinalcord compression.

Perivascular storage of lysosomal storage granules around the vessels ofthe brain may produce cysts. Such cysts and the effectiveness of thetherapeutic regimens of the present application against such cysts mayalso be assessed using MRI scans to determine the size and number ofsuch cysts. Any reduction in size and/or number of the cysts will beconsidered to be a therapeutically beneficial effect of the methods ofthe present invention.

Any release or reduction in the CSF pressure, reduction in the sizeand/or number of cysts or any other decrease in the symptoms caused bythe presence of lysosomal storage granules as a result of thetherapeutic regimens of the present invention will be considered to be atherapeutic benefit of the present invention. Such decreases arepreferably in the order of at least 5% as compared to the levels of suchsymptoms prior to the administration. Of course, a greater decrease,e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or more would bepreferable. Most preferably, the symptoms are reduced/ameliorated tosuch an extent as to make the symptoms in subject indistinguishable fromthe same indicia observed in a normal healthy subject of like sex, ageand physical characteristics.

Assessment of Methods in Model Animals

The methods of the present invention may be evaluated using models oflysosomal storage disease that are known to those of skill in the art.For example, a canine model of MPS I may be used as described in theexamples herein below. Other models of MPS also may be used. Manypre-clinical studies rely on mouse models for a given disease. One suchmodel is the MPS model described in example 1 of U.S. Pat. No.6,582,692, which details crossing of Gus^(mps/+) mice in the originalC57BL6 (B6) background (Jackson Laboratory, Bar Harbor, Me., USA) withthe congenic strain B6Gusa (Pfister et al. (1982) Biochem Genet 20:519-535) to produce Gus^(mps/a) progeny for a breeding colony in whichboth parents were always Gus^(mps/a) and progeny carried mps/a, a/a ormps/mps allele combinations. The parameters for GUS activity may bemonitored as described in that patent.

Yet another model that may be useful for evaluating the methods of thepresent invention is one exemplified in U.S. Pat. No. 6,002,067, whichis transgenic mouse model for iduronidase deficiency. Of course, thoseof skill in the art also will be aware of other models that may be usedin evaluating the methods of the present invention. Once the methodshave been evaluated in such model animals, the methods are then readilyscaled and adapted for the treatment of other mammalian subjects such asprimates and preferably human subjects.

One of the most dramatic characteristics of MPS disease is theappearance of large, cytoplasmic, storage vacuoles apparent bymicroscopic examination of tissue sections (Vogler et al. (1990) Am JPathol 136: 207-217). In one example using a MPS mouse model, MPS miceinjected intrathecally with a composition comprising either iduronidaseor saline alone are sacrificed 4 weeks after administration and examinedhistologically, as follows. Mice are killed by cervical dislocation andimmediately perfused via the left ventricle, first with saline and thenwith 10% neutral buffered formalin. The fixed animals with exposedviscera are then immersed in formalin before histopathologic evaluation.Selected tissues are processed using routine techniques, embedded inparaffin, cut at approximately 5 microns, stained with hematoxylin andeosin, and examined microscopically. In particular, it will be desirableto perform such histopathologic evaluation on meningeal and/or neuronalcells of the animals.

Brain sections from control MPS animals should show moderate to severe,diffuse, cytoplasmic vacuolations in meninges and/or neuronal cells.Such cells from treated mice have a substantial reduction of storagevacuoles resulting in normal tissue architecture.

In order to assess the presence of storage granules in brain, mice aresacrificed by cervical dislocation, the brain was removed and onehemisphere was fixed in 10% neutral buffered formalin. 5 μM sections arestained with hematoxylin and eosin (H and E).

As discussed above, both neonatal and adult animals may be treated withintrathecal administration of iduronidase. In model mice, three-day oldneonatal mice and e.g., adult mice of 7-13 weeks old at the time ofinjection can be treated with 0.01 μg to about 5 μg enzyme. It should benoted that dose is likely to the 1/1000^(th) the dose that required fora larger mammal such as a dog. For intrathecal administration tonewborns, mice are anesthetized by inhalation of halothane, andiduronidase in 30 μl saline (with 2% dye) may be injected between thesixth lumbar and second sacral vertebrae using a 30-gauge needle.Successful introduction into the cerebrospinal fluid space is detectedimmediately as a green streak extending from the spinal column anddiffusing into the brain. For intrathecal administration to adults, MPSmice are anesthetized with avertin (tribromoethanol) and a 1 cm incisionis made through the skin parallel to the spine to make the positions ofindividual vertebrae visible. Iduronidase with 2% dye may then beinjected between the last thoracic and second lumbar vertebrae.

At increasing times after this treatment, mice are sacrificed andtissues analyzed for iduronidase levels. Therapeutic levels ofiduronidase enzyme are scored as any level which produced a detectabledecrease in storage vacuoles. Of course, the above model studies arepresented merely by way of example, with other exemplary model studiesare described in the Examples herein below, these model studies mayreadily be modified without departing from the scope of the claimedinvention.

Modification of Enzyme to Facilitate Improved Uptake

In the methods of the present invention, it may be preferably to ensurethat the enzyme being administered to the subject through intrathecaladministration is one which comprises a moiety that may be readily takenup by a high affinity uptake receptor on the surface of a brain cells.For example, such a receptor may be the mannose-6-phosphate receptor andthe enzyme comprises up to about an average of about at least 20%bis-phosphorylated oligosaccharides per enzyme. In other embodiments,the enzyme may comprise 10%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%bis-phosphorylated oligosaccharides per enzyme. While suchbis-phosphorylated oligosaccharides may be naturally present on theenzyme, it should be noted that the enzymes may be modified to possesssuch oligosaccharides. For example, those of skill in the art are awareof enzymes which are capable of catalyzing the transfer ofN-acetylglucosamine-L-phosphate from UDP-GlcNAc to the 6′ position ofα-1,2-linked mannoses on lysosomal enzymes. Methods and compositions forproducing and using such enzymes are described by, for example, Canfieldet al. in U.S. Pat. No. 6,537,785, and U.S. Pat. No. 6,534,300, eachincorporated herein by reference.

In other embodiments, the lysosomal enzymes for use in the presentinvention may be conjugated to a RAP and RAP polypeptides, whichselectively bind to LRP receptors that may be present on brain cells. Assuch, these RAP molecules will serve to increase the transport of thelysosomal enzyme across the blood brain barrier and/or deliver agents tolysosomes of cells within the CNS. Methods and compositions forpreparing enzyme compositions that comprise RAP moieties attachedthereto are described in detail in U.S. patent application Ser. No.10/206,448, filed on Jul. 25, 2002 and in U.S. patent application Ser.No. 10/600,862, filed Jun. 20, 2003, each incorporated herein byreference.

In still a further embodiment, those of skill in the art may employ adelivery of the enzyme conjugated to melanotransferrin (p97) asdescribed in e.g., U.S. Pat. No. 6,455,494 and U.S. Pat. No. 5,981,194,each incorporated herein by reference. Of course the above agents thatenhance the delivery and/or uptake of therapeutic agents to brain tissueare merely exemplary and those of skill in the art will be aware ofother receptors, ligands or other agents that may be used in a similarcontext to deliver a therapeutic agent across the brain-CSF interface oreven the BBB.

Combination Therapy to Tolerize Subject to Enzyme Replacement Therapy

It has been found that during administration of agents such asrecombinant proteins and other therapeutic agents, a subject can mountan immune response against these agents, leading to the production ofantibodies that bind and interfere with the therapeutic activity as wellas cause acute or chronic immunologic reactions. This problem is mostsignificant for therapeutics that are proteins because proteins arecomplex antigens and in many cases, the subject is immunologically naïveto the antigens. Thus, in certain aspects of the present invention, itmay be useful to render the subject receiving the therapeutic enzymetolerant to the enzyme replacement therapy. In this context, the enzymereplacement therapy may be given to the subject as a combination therapywith a tolerizing regimen.

Co-owned, co-pending U.S. patent application Ser. No. 10/141,668(incorporated herein by reference) discloses treatment of lysosomalstorage disorders using immune tolerance induction. Briefly, use of sucha tolerization regimen may be useful to prevent the subject mounting animmune response to the enzyme replacement therapy and thereby decreasingor otherwise rendering ineffective the potential beneficial effects ofthe enzyme replacement therapy.

In a preferred method, the invention contemplates reducing or preventinga clinically significant antigen-specific immune response to recombinanthuman α-L-iduronidase used to treat mucopolysaccharidosis I (MPS I),where the iduronidase is administered intrathecally. The method employsan initial 30-60 day regimen of a T-cell immunosuppressive agent such ascyclosporin A (CsA) and an antiproliferative agent, such as,azathioprine (Aza), combined with weekly intrathecal infusions of lowdoses of iduronidase. The typical strong IgG response to weeklyinfusions of iduronidase becomes greatly reduced or prevented using a 60day regimen of immunosuppressive drugs, cyclosporin A (CsA) andazathioprine (Aza), combined with weekly intrathecal infusions of lowdoses of rhIDU. Using such tolerization regimens, it will be possible torender the subject tolerant to higher therapeutic doses of iduronidasefor up to 6 months without an increase in antibody titer against theiduronidase, or indeed any other enzyme that could be used for enzymereplacement of a lysosomal storage disease. Such tolerization regimenshave been described in U.S. patent application Ser. No. 10/141,668,specifically incorporated herein by reference.

Intrathecal Administration of the Pharmaceutically AcceptableFormulations

As discussed above, the present invention is based on surprisingdiscoveries of the therapeutic efficacy of using intrathecaladministration of enzyme replacement therapy for lysosomal storagedisease. In one embodiment, the enzyme is administered by introductioninto the central nervous system of the subject, e.g., into thecerebrospinal fluid of the subject. In certain aspects of the invention,the enzyme is introduced intrathecally, e.g., into the lumbar area, orthe cistema magna or intraventricularly into a cerebral ventricle space.

Those of skill in the art are aware of devices that may be used toeffect intrathecal administration of a therapeutic composition. Forexample, the therapy may be given using an Ommaya reservoir which is incommon use for intrathecally administering drugs for meningealcarcinomatosis (Lancet 2: 983-84, 1963). More specifically, in thismethod, a ventricular tube is inserted through a hole formed in theanterior horn and is connected to an Ommaya reservoir installed underthe scalp, and the reservoir is subcutaneously punctured tointrathecally deliver the particular enzyme being replaced, which isinjected into the reservoir. Other devices for intrathecaladministration of therapeutic compositions to an individual aredescribed in U.S. Pat. No. 6,217,552, incorporated herein by reference.Alternatively, the drug may be intrathecally given, for example, by asingle injection, or continuous infusion. It should be understood thatthe dosage treatment may be in the form of a single dose administrationor multiple doses.

As used herein, the term “intrathecal administration” is intended toinclude delivering a pharmaceutical composition directly into thecerebrospinal fluid of a subject, by techniques including lateralcerebroventricular injection through a burrhole or cistemal or lumbarpuncture or the like (described in Lazorthes et al. Advances in DrugDelivery Systems and Applications in Neurosurgery, 143-192 and Omaya etal., Cancer Drug Delivery, 1: 169-179, the contents of which areincorporated herein by reference). The term “lumbar region” is intendedto include the area between the third and fourth lumbar (lower back)vertebrae and, more inclusively, the L2-S1 region of the spine. The term“cisterna magna” is intended to include access to the space around andbelow the cerebellum via the opening between the skull and the top ofthe spine. The term “cerebral ventricle” is intended to include thecavities in the brain that are continuous with the central canal of thespinal cord. Administration of a pharmaceutical composition inaccordance with the present invention to any of the above mentionedsites can be achieved by direct injection of the composition or by theuse of infusion pumps. For injection, the composition of the inventioncan be formulated in liquid solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution orphosphate buffer. In addition, the enzyme may be formulated in solidform and re-dissolved or suspended immediately prior to use. Lyophilizedforms are also included. The injection can be, for example, in the formof a bolus injection or continuous infusion (e.g., using infusion pumps)of the enzyme.

In one embodiment of the invention, the enzyme is administered bylateral cerebro ventricular injection into the brain of a subject. Theinjection can be made, for example, through a burr hole made in thesubject's skull. In another embodiment, the enzyme and/or otherpharmaceutical formulation is administered through a surgically insertedshunt into the cerebral ventricle of a subject. For example, theinjection can be made into the lateral ventricles, which are larger,even though injection into the third and fourth smaller ventricles canalso be made.

In yet another embodiment, the pharmaceutical compositions used in thepresent invention are administered by injection into the cisterna magna,or lumbar area of a subject.

In another embodiment of the method of the invention, thepharmaceutically acceptable formulation provides sustained delivery,e.g., “slow release” of the enzyme or other pharmaceutical compositionused in the present invention, to a subject for at least one, two,three, four weeks or longer periods of time after the pharmaceuticallyacceptable formulation is administered to the subject.

As used herein, the term “sustained delivery” is intended to includecontinual delivery of a pharmaceutical composition of the invention invivo over a period of time following administration, preferably at leastseveral days, a week or several weeks. Sustained delivery of thecomposition can be demonstrated by, for example, the continuedtherapeutic effect of the enzyme over time (e.g., sustained delivery ofthe enzyme can be demonstrated by continued reduced amount of storagegranules in the subject). Alternatively, sustained delivery of theenzyme may be demonstrated by detecting the presence of the enzyme invivo over time.

The pharmaceutical formulation used in the method of the inventioncontains a therapeutically effective amount of an enzyme for use inenzyme replacement therapy of a lysosome storage disease. Such atherapeutically effective amount is any amount effective, at dosages andfor periods of time necessary, to achieve the desired result. Inpreferred embodiments, the compositions comprises a therapeuticallyeffective amount of iduronidase. A therapeutically effective amount ofiduronidase may vary according to factors such as the disease state,age, and weight of the subject, and the ability of the enzyme (alone orin combination with one or more other agents) to elicit a desiredresponse in the subject. Dosage regimens may be adjusted to provide theoptimum therapeutic response. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the composition areoutweighed by the therapeutically beneficial effects. A non-limitingrange for a therapeutically effective concentration of iduronidase is0.001 μg enzyme/ml to about 150 μg enzyme/ml. It is to be noted thatdosage values may vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the enzymereplacement therapy and that dosage ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed invention.

The enzyme composition is preferably in the form of an injectable unitdose. Examples of carriers or diluents usable for preparing suchinjectable doses include diluents such as water, ethyl alcohol,macrogol, propylene glycol, ethoxylated isostearyl alcohol,polyoxyisostearyl alcohol and polyoxyethylene sorbitan fatty acidesters, pH adjusting agents or buffers such as sodium citrate, sodiumacetate and sodium phosphate, stabilizers such as sodium pyrosulfite,EDTA, thioglycolic acid and thiolactic acid, isotonic agents such assodium chloride and glucose, local anesthetics such as procainehydrochloride and lidocaine hydrochloride. Furthermore usualsolubilizing agents and analgesics may be added. Injections can beprepared by adding such carriers to the enzyme or other active,following procedures well known to those of skill in the art. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

The pharmaceutically acceptable formulations can easily be suspended inaqueous vehicles and introduced through conventional hypodermic needlesor using infusion pumps. Prior to introduction, the formulations can besterilized with, preferably, gamma radiation or electron beamsterilization.

Kits for Use in the Methods of the Invention

The agents utilized in the methods of the invention may be provided in akit, which kit may further include instructions for use. Such a kit willcomprise an enzyme for use in the treatment of a lysosomal storagedisease, usually in a dose and form suitable for administration to thehost. The kit will usually comprise a device for delivering the enzymeintrathecally. The kit may further comprise a T cell immunosuppressiveagent, in a form suitable for administration, and may further includeassay reagents for monitoring blood levels of the agent, and/or fordetermination of suppression of T cell activity. An anti-proliferativeagent may also be included, in a form suitable for administration.

A kit may also provided for the conjugation of an antigen, particularlya polypeptide antigen, to a high uptake moiety, in order to generate atoleragenic composition. For example, a moiety such as a mannose 6phosphate group, either conjugated to a linker suitable for linkingsugars and polypeptides, as described above, may be provided. The highuptake moiety may also be provided in an unconjugated form, incombination with a suitable linker, and instructions for use.

Another kit may comprise instructions for the intrathecal administrationof the therapeutic compositions of the present invention, in addition tothe therapeutic compositions. In certain embodiments, the kits of theinvention may comprise catheters or other devices for the intrathecaladministration of the enzyme replacement therapy that are preloaded withthe therapeutic compositions of the present invention. For example,catheters preloaded with 0.001 mg, 0.005 mg, 0.01 mg, 0.015 mg, 0.02 mg,0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg,0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1.0mg or more of iduronidase in a pharmaceutically acceptable formulationare specifically contemplated. Other enzymes for use in lysosomalstorage diseases also may be similarly presented in preloaded cathetersfor intrathecal administration. Exemplary catheters may single usecatheters that can be discarded after use. Alternatively, the preloadedcatheters may be refillable and presented in kits that have appropriateamounts of the enzyme for refilling such catheters.

Additional aspects and details of the invention will be apparent fromthe following examples, which are intended to be illustrative ratherthan limiting.

Example 1 Protocols for Assessing Direct Injection of the Brain withRecombinant Human Iduronidase

Those of skill in the art also are aware of well-known canine models forlysosomal storage diseases. In one embodiment, MPS I canines are used toassess the efficacy of the methods of the present invention. For suchdeterminations, it is desirable that normal canines and MPS I caninesare assessed concurrently. The following example provides exemplaryprotocols for use in conjunction with the methods described herein.

In order to assess enzyme penetration in the brain of normal canines,normal beagle dogs (e.g., 2 initially planned, up to 4 possible) areprepared for anesthesia and sterility. An Ommaya reservoir or equivalentdevice is implanted with a ventricular catheter placed in the lateralventricle. A CSF reservoir and lumbar catheter may also be implanted inthe lumbar region. CSF is withdrawn to confirm patency. Enzymeadministration and CSF sampling is performed at the lateral ventricle inone of the beagles. The system at the lumbar region in this beagleserves as a backup system in the event of irreversible problems occursat the original site of access. The second beagle is set up to receiveenzyme and CSF sampling at the lumbar region. The system at the lateralventricle in this second beagle will serve as a backup system in theevent of irreversible problems at the original site of access. Theenzyme is administered at weekly injections for four weeks. PK studiesof CSF clearance of iduronidase are assessed using a set of timedsamplings at the first and last week of injection. All CSF samplesobtained are analyzed for safety, PK and pharmacodynamics of enzymepenetration. In the event that complications arise while placing theventricular system in the normal beagles, the methods of the presentinvention may be assessed using an administration system placed only inthe lumbar region. Thus, enzyme administration and CSF sampling willoccur at the lumbar regions only. At the termination of treatment, braintissue may be collected to assess iduronidase activity using a validatedassay. Brain tissue will also analyzed for storage using lightmicroscopy and confocal immunofluorescence. Tissues both proximal anddistal to the site of ventricular penetration may be assessed for enzymepenetration.

In order to determine the enzyme penetration in the model animals forlysosomal storage disease, the above protocol is repeated using MPS Icanines.

To assess the response of the animals to the treatment, a variety ofparameters may be monitored. To obtain a baseline assessment, it may bedesirable to perform a clinical examination to assess, physicalcondition, vital signs and weight. This assessment should preferably besupplemented with clinical laboratory analyses to determine the CompleteBlood Count (CBC) and Superchem profile and urinalysis of the animals.Urine specimens should be analyses for the presence ofglycosaminoglycans, serum analysis should be performed to assess thepresence of anti iduronidase antibodies as this may have an affect onthe amount of iduronidase that should be administered. Plasmaiduronidase activity also should be assessed. The baseline assessmentalso should include an analysis of the CSF for standard CSF lab analysis(cell count, protein, glucose and cytology), GAG, ELISA for anti-iduantibodies, and iduronidase analysis. The cells present in the CSFshould be assessed for the presence of storage granules using a simplestain. These parameters should then be assessed periodically throughoutthe period of therapy. At the end of the analysis period, brain tissuemay be obtained and analyzed further. Such analyses may include a brainbiopsy to perform an iduronidase assay and tissue GAG levels. Thepathology of MPS I animals may be assessed using light and electronmicroscopy, and confocal immunofluorescence using anti-iduronidaseantibodies also may be performed.

The following is a discussion of the general methods used to perform theabove-outlined assessments.

Clinical Examination:

In order to assess the physical condition of the animals, a generalphysical examination should note the posture, activity, demeanor andgeneral appearance, preferably on a daily basis throughout the course ofthe experiment the examination should note the vital signs of the animal(heart rate, body temperature, and respiratory rate), particularly aftereach injection. Growth may be assessed by periodically taking bodyweight measurements.

CSF and Plasma α-L-Iduronidase Levels:

Enzyme levels may be measured in the plasma and CSF just prior to enzymeadministration to the CSF each week. CSF is obtained after sterilepreparation of the CSF port and accessed with a sterile needle. Theenzyme in blood samples is stabilized by adding 1/10^(th) volume of 100mM NaPO4/Citrate pH 4.0 The enzyme is assayed for iduronidase using avalidated assay with the artificial substrate4-methylumbelliferyl-α-1-iduronide. Net fluorescence is determined byfluorometry at 365 nm excitation and 440 nm emissions. One unit ofiduronidase is equivalent to the number of micromoles of substratecleaved per minute at 37° C. in the conditions of the assay.

Brain Tissue α-L-Iduronidase:

Enzyme levels may be measured in a biopsy specimen obtained. In the MPScanines, biopsies may be obtained prior to perfusion. A brain sample issnap frozen in a labeled vial in liquid nitrogen. After thawing, thespecimen is weighed quickly and 3 vol of PAD (10 mM phosphate-buffer pH5.8, 0.02% azide and 0.1 mM dithiothreitol)+0.1% Triton X-100. Thetissue sample is ground in Dounce ground glass homogenizer by a minimumof 10 strokes while on ice and the homogenate cleared of large particlesby spinning in a microfuge for a few seconds. The extract is stored bysnap freezing. The enzyme is assayed for iduronidase using a validatedassay with the artificial substrate 4-methylumbelliferyl-α-1-iduronide.Pilot assays should preferably be performed to determine the time ofassay required and whether dilution is needed. Net fluorescence isdetermined by fluorometry at 365 nm excitation and 440 nm emissions. Oneunit of iduronidase is equivalent to the number of micromoles ofsubstrate cleaved per minute at 37° C. in the conditions of the assay.

GAG Analyses:

At the conclusion of the therapy, the dogs may be euthanized and braintissue samples collected by biopsy and quick frozen with liquid nitrogenfor subsequent tissue glycosaminoglycan analysis. For tissue GAGanalysis, sulfated glysocaminoglycans will be assayed using amodification of the Alcian Blue method of Bjornsson as published(Kalckis et al., Biochem Mol Med. 1996; 58(2):156-67). The GAGquantities can be determined by comparison to standards of dermatansulfate. Urinary and CSF GAG quantification is completed in a methodnearly identical to that used to quantify tissue GAG content performedon urine and CSF samples.

CSF Storage:

Cellular debris from the CSF can be identified using a simple stain andthe cells readily assessed for GAG storage.

CSF Pharmacokinetics Studies:

Pharmacokinetic studies may be completed on each treated dog during thefirst and last weeks of enzyme replacement therapy to monitorα-L-iduronidase clearance from the CSF following an infusion. Afteradministration of enzyme to the CSF via the ventricular port, samplesare drawn from the same site of enzyme administration at 1, 2, 4 hours.The samples are withdrawn and prepared as in the section on CSF samples.Data is plotted as time versus CSF iduronidase activity. Half-life ofiduronidase in the circulation can be determined.

CBC, Superchem Profile and Urinalysis:

Blood samples are collected every two weeks for a CBC and superchemprofile. Urinalysis with reagent strips is also performed every otherweek on a fresh urine sample to monitor items such as proteinuria andhematuria.

ELISA for α-L-Iduronidase Specific Antibodies:

Serum samples are collected and frozen at −20° C. for subsequentantibody analysis. Antibodies specific for iduronidase are detected bystandard ELISA protocol using goat anti-dog IgG labeled with alkalinephosphatase as the secondary antibody. Antibodies in the CSF aredetermined by the same method though it is expected that a smallerdilution may be needed.

Enzyme Composition Delivered:

Recombinant human α-L-iduronidase is supplied by BioMarin Pharmaceuticalfrom bulk lots that may or may not be released for human use. The enzymeshould preferably meet all relevant specifications required for enzymetherapy and safe administration including passing potency, activity,sterility, and endotoxin levels. The dosage form consists of enzyme at100,000 u/ml in formulation buffer (100 mM NaPO4, pH 5.8, 150 mM NaCl atpH 5.3-5.8).

Placement of the Indwelling Ventricular Device:

Procedures have been described for sampling via the ventricular system(McCully et al; Poplack et al; Moir and Dow et al; Kusumi and Plouffe;Haslberger and Gaab). Some of these also involve trauma to the brain anddo not permit precise positioning of the delivery system. We will use atechnique that permits an investigator to obtain multiple sterile CSFsamples or administer multiple injections into the CSF ofunanaesthetized animals that are restrained with a minimal dose oftranquilizers. The procedure involves the placement of an indwellingcatheter into the lateral ventricles as well as the intrathecal space oflumbar region of the spine.

In the examples discussed herein, the animals, e.g., two normal, male,adult laboratory-reared Beagle dogs and two, male dogs withmucopolysaccharidosis I are used. Dogs are atropinized (0.045 mg/kg),and anesthesia is induced with intravenous Propofol (1-6 mg/kg) titratedto effect. The dogs are intubated and maintained on Isofluraneanesthesia with oxygen, and placed on a heating pad during surgery tomaintain normal body temperature. Normal saline will be administered forfluid maintenance. Antibiotics may be administered prior to and duringsurgery to prevent infection.

The dog is placed on its ventrum with the head supported to ensure thatthe airway remains patent. The occiput and dorsal midline is clipped,surgically scrubbed and draped. Sterile technique and loupemagnification is used throughout the procedure. The appropriate lengthof the catheter is predetermined by measuring the thickness of the firsttwo cervical vertebrae (C1 and C2), the distance from C2 and thedistance to the cistern. The volume of fluid necessary to fill thevolume of the catheter and reservoir (dead space) is calculated.

The skin is incised on the midline from the occipital prominence alongthe dorsal midline to expose the foramen magnum, the junction of C1 andthe occiput, and the atlanto-occipital membrane. The subcutaneousmuscles are sharply dissected and the ligamentum nuchae are divided.Using an air drill and scalpel a small keyhole is created in theposteruir ekenebts of C1 and a 2 mm horizontal slit is made in the durato enter the cisterna magna, Using a surgical hook, the pre-measuredlength of a perforated Spetzler lumbar silicone catheter containing astylet is threaded into the ventricle space and CSF is withdrawn toconfirm patency. The catheter is anchored to the muscle near thereservoir. Hemostasis is accomplished with a bipolar electrocauteryunit. A subcutaneous subgaleal pocket is created in the occipital areato accommodate the Ommaya reservoir. The reservoir is secured withnon-absorbable suture to the occipital pericranium. The remainingexternal portion of the catheter is extended to the subcutaneous pocket,and a metal step-down connector is used to attach the catheter to theOmmaya reservoir and silk suture may be used to ensure the connection.

To determine the patency of the catheter, a small quantity of CSF, whichjust exceeds the combined dead space of the catheter and reservoir, isgently withdrawn using a 25-gauge ⅝-inch needle affixed to a 1 ccsyringe. The reservoir is secured with non-absorbable sutures to theoccipital pericranium. The system is examined for leakage, and theoperative site closed in anatomical layers with interrupted 3.0 Vicrylsutures. The skin is then closed with nylon sutures.

Withdrawal of CSF or injections into the reservoir are done usingsterile technique (surgical scrub of the skin and sterile gloves) with a25 gauge ⅝ inch needle affixed to a 1 cc syringe. The CSF should bewithdrawn gently and steadily.

Postoperatively, the dog is monitored and intravenous fluids areadministered as needed until it is able to stand, eat and drink. Theanalgesic buprenorphine (0.01 mg/kg SQ at 12 hour internals) may beadministered as necessary to relieve discomfort. Antibiotics may beadministered 10 days postoperatively. The dog should be clinicallyexamined daily.

To maintain patency, the system should be flushed weekly. This willallow sampling of CSF and administration of enzyme. To administer theenzyme or withdraw CSF, the dogs are restrained with 0.1 mg/kg ofacepromazine and increased as needed. The skin over the reservoir isclipped and surgically scrubbed. Wearing sterile gloves, the location ofthe reservoir behind the ear is determined, and entered using a 25-gaugeneedle attached to a 1 ml syringe. Particular care should be taken toenter the dome of the reservoir a few millimeters away from the area ofthe skin puncture to minimize exogenous contamination. A volume of CSFequal to the volume of the catheter plus the reservoir is removed anddiscarded by removing the syringe and expelling its contents; then, thedesired amount of CSF is withdrawn or the enzyme administered into thereservoir. When enzyme is administered (18,000 units/mL CSF), it ispreferable to “chase” the enzyme with a volume of physiologic salineequivalent to the dead space volume of the catheter and reservoir. Thisensures that the enzyme is administered directly into the ventricularsystem. CSF sampling and enzyme administration is continued as discussedabove following installation of the catheter.

For tissue analysis, after eight weeks of enzyme treatment the dogs aredeeply anesthetized (loss of toe pinch and eyelid reflexes) with anoverdose of sodium pentobarbital, and flushed intracardially withheparinized saline. A small cranial opening is made in the frontal areaand a small section of brain is removed. The dog is then perfusedintracardially with 4% paraformaldehyde.

Throughout the above treatment protocols, the canines should bemonitored closely for signs of an anaphylactic reaction during andimmediately after enzyme administration. Signs of a reaction may includebehavioral changes, such as restlessness, irritability, or extremestillness, as well as vomiting, bowel movements, and loss of color inthe mucous membranes. If any of these symptoms or other adverse symptomsoccur, the administration should be stopped, diphenhydramine may beadministered, followed by a saline drip and administration of oxygen.The infusion may be continued when the reaction subsides.

Canines are also monitored for infection due to the exteriorizedcatheter and are treated by appropriate measures (catheter removal,local control of infection, systemic antibiotics). If an infectionoccurs, such as ventriculitis, enzyme treatments will be postponed untilthe infections has been adequately treated with gentamicin.

The following examples describe the results of studies performed on theintrathecal administration of iduronidase to MPS I model animals usingsome or all of the methods described in the above example.

Example 2 Enzyme Administered Via Intraventricular Injection PenetratesBlood Brain Barrier and is Detected in Brain Tissue

Administration of enzymes directly to the site of lysosomal storageinduced damage in the brains of subjects with lysosomal storage disorderhas proven difficult to this point. The large enzyme complexes necessaryto treat these diseases typically cannot penetrate the blood brainbarrier. To determine an effective method for drawing these enzymesacross the brain-CSF interface, two routes of enzyme administration weretested in a rat and canine model of the lysosomal storage disorder MPSI.

To administer enzyme intraventricularly, rats were injected in thelateral ventricle, using sterotactic guidance, with either 5-10 μl ofrecombinant human iduronidase (rhIDU) or control protein. Animals weresacrificed 24 hours after injection and brain sections obtained.

Brain sections were analyzed for the presence of rhIDU using confocalmicroscopy with anti-iduronidase antibodies. Immunohistochemicalanalysis showed that the injected enzyme is taken up by brain neurons,and further that the iduronidase is localized to the lysosomes in theneuronal cells. Anti-IDU staining indicates that the enzyme penetratesthe brain tissue for several millimeters, but there is a decreasinggradient of enzyme, meaning that the farther away from the injectionsite the less enzyme is detected in the brain. The staining alsoindicated that the half-life of the enzyme was approximately 7 days.

Brain sections were also analyzed for rhIDU activity.

Example 3 Enzyme Administered Via Intrathecal Injection Penetrates andis Detected in Brain Tissue

To determine whether intrathecal injection of enzyme involved inlysosomal storage disorders could cross the blood brain barrier aseffectively, or more effectively, than intraventricular injection,intrathecal injection into the CSF was performed in canine subjects.

Animals (n=2/group) were administered 1 cc rh-iduronidase, with a totalprotein content of 0.33 mg, 1 mg, or 3 mg, via injection into thecisterna magna. This protocol was repeated weekly for a total of fourweeks. Brain sections were taken for analysis 48 hours after the lastinjection. For analysis, the right half of the brain was slicedcranially and alternate sections analyzed for enzyme activity,immunohistochemical localization of enzyme in brain andglycosaminoglycan content in brain sections. The left half of the brainwas sliced coronally and assayed by light microscopy and electronmicroscopy.

Analysis of enzyme levels in brain of subjects after intrathecalinjection (FIG. 1) demonstrated that animals given 0.33 mg ofiduronidase show 5-fold increase of enzyme in the brain compared tocontrol animals (mean, 65±28 Units/mg protein), animals receiving 1mg/injection showed a 7-fold increase in enzyme (mean enzyme levels of89±62 U/mg), while animals receiving 3 mg enzyme/injection showed a17-fold increase in enzyme levels, with a mean iduronidase level ofapproximately 224±32 U/mg. Thus, increasing the dosage of iduronidaseadministered to a subject increases the level of iduronidase in thedetected in the brain.

In similar experiments in which the 6 dogs were treated with low (0.46mg/injection), medium (1.08/1.38 mg/injection) and high (4.14mg/injection) of rh-iduronidase doses administered via the cistema magnaonce per week for four weeks and assayed the iduronidase content of thebrain at 48 hours after the last dose (see Table 1 for specific doses).The two intrathecally-treated dogs at each dose level were compared withthe iduronidase enzyme levels in two untreated normal dogs. TheIT-treated dogs had 5.6, 7.5 and 18.9-fold the enzyme levels ofuntreated or vehicle-treated animals for the low, medium, and highdoses, respectively (Table 1). Given that the corrective concentrationof enzyme is as low as 2-5% of normal, these levels representedconcentrations very far above the required corrective levels of enzyme.

TABLE 1 Dose-response effects of rhIDU administered IT to normal dogsWeekly Dose of IT rhIDU (mg) Total Brain ^(¥) Fold Normal Surface Brain^(¥) Fold Normal Deep Brain ^(¥) Fold Normal Untreated/ 11.9 ± 1.95 1 ND1 ND 1 Placebo- [10.1-15.0] treated Normal Low (0.46) 66.4 ± 4.07 5.6101 ± 19.5  8.5 31.8 ± 3.75 2.7 [63.5, 69.3] p = 0.0001* [87.5, 115] p =0.0001* [29.1, 34.4] p = 0.0002* Medium 89.0 ± 18.2 7.5 121 ± 43.4 10.252.3 ± 0.64 4.4 (1.08/1.38) [76.0, 102]  p = 0.0001* [90.5, 152] p =0.0011* [51.8, 52.7] p < 0.0001* High (4.14)  225 ± 89.5 18.9  355 ±198  29.8 70.6 ± 14.1 5.9 [161, 288] p = 0.0014*  [214, 495] p = 0.0057*[60.6, 80.5] p = 0.0001* Iduronidase levels are calculated from meanvalues for each region for each dog sacrificed. Means of the means foreach animal ± standard deviation are shown. N = 5 for the untreatedgroup and N = 2 for each dosage group. ^(¥) Iduronidase levels areexpressed in units of iduronidase per mg protein. *Statisticallysignificant. ND is not done.

Levels of rh-iduronidase were also measured in deep brain and surfacebrain tissues (FIG. 2) of animals given either 0.33 mg, 1 mg or 3 mgenzyme. Again, analyses showed that the higher the dose of enzyme thegreater the amount of iduronidase detected in the brain tissue, with the3 mg/injection group demonstrating a 5-fold increase in deep braintissue. Iduronidase measured on the surface of brain tissue was detectedat a 8-fold difference in animals receiving 0.33 mg protein/injectionwhile animals receiving 3 mg/injection exhibited a 27-fold increase insurface expression of iduronidase compared to normal controls. Thus,while the majority of iduronidase is detected on the surface braintissue, a significant amount penetrates into deep brain tissue,indicating this type of treatment would be a useful therapy foradministration of enzymes in lysosomal storage disorders of deep braintissue. Additional experiments in which the low, medium and high doseswere 0.46 mg; 1.08/1.38 mg; and 4.14 mg, respectively, showed that deepbrain specimens had 2.7, 4.4 and 5.9-fold of normal activity at theserespective doses.

Immunohistochemical analysis by confocal microscopy showed that largeamounts of rh-iduronidase could be detected on the surface of the cortexas well as inside cells of the hippocampus. Particularly, glial cells inthe hippocampus, the part of the brain involved in memory, take upsignificant amounts of enzyme. Staining also demonstrated that theenzyme diffuses into the brain and some glial cells stain brightly withanti-iduronidase. The higher doses do not result in substantially higherα-L-iduronidase activity in the deep regions of the brain and hence, adose of approximately 1 mg was selected for treating MPS I dogs in thefurther studies.

These results indicate that intrathecal injection of rh-iduronidaseprovides an efficient method for administering protein across the bloodbrain barrier. The protein is detectable both on the surface of braincells and in lysosomes of brain cells, as shown in Example 2,demonstrating that intrathecal injection is an effective means fortransporting enzymes involved in lysosomal storage disorders can beadministered intrathecally and provide a therapeutic benefit to subjectsaffected by said disease.

Example 4 Intrathecal Injection of Rh-Iduronidase Ameliorates MPS ISymptoms

Initial experiments demonstrated that iduronidase administered viaintrathecal injection effectively crossed the blood brain barrier andcould be detected in significant amounts in the lysosomes of neurons, onthe surface of cerebral cortex cells, and also penetrated into deepbrain tissue. Based on these results, it seems likely that lysosomalstorage disorders that impair brain function could be treated viaintrathecal injection of enzyme replacement therapy.

To assess the efficacy of intrathecal injection of enzymes involved inlysosomal storage disorders, canine subjects affected with the lysosomalstorage disorder MPS I and lacking the enzyme iduronidase were treatedwith an intrathecal administration of rh-iduronidase. The iduronidaselevels in the brain and central nervous system tissue assessed after 4weeks of treatment.

As noted above, a dose of 1 mg rh-iduronidase/injection was selected.Four MPS I affected animals were treated with 1 mgrh-iduronidase/injection via intracisternal injection, one time per weekfor four weeks and enzyme levels measured 48 hours after the lasttreatment dose. The intrathecal injections resulted in widespreaddistribution of the enzyme in the brain, spinal cord, and meninges.Detection of enzyme activity in MPS I animals revealed a mean 21-foldincrease in iduronidase levels in these animals compared to the controlgroup. Analysis of enzyme activity in deep brain and surface braintissue of MPS I animals showed an average of 11-fold and 37-foldincrease in activity, respectively.

In a further set of experiments, the overall brain enzyme activity inthe four treated dogs reached a mean 277 units/mg compared with a meanlevel of 11.9 units/mg in untreated normal dogs, and averaged 23-foldnormal with a range of 17-34 fold normal levels. As was the case for thenormal dogs, α-L-iduronidase activities were higher (3-4 fold) at thesurface of the brain than its internal regions (474.0±257.7 vs.138.7±93.5). Nevertheless, the levels in deep brain were still over 11times normal.

Because intrathecal administration of a protein places the proteindirectly into the CSF, which bathes the entire central nervous system,it is likely that any protein injected via this route is detectable inall areas of the CNS. MPS I animals used above were used to assess thepresence of iduronidase in the spinal cord and meninges of treatedanimals as described above in Example 1.

Spinal cord and meninges samples were obtained from four MPS I animalsand iduronidase activity measured as above (mean of cervical, thoracicand lumbar regions). Spinal cord levels of iduronidase activity in MPS Ianimals was on average 13-fold higher than control animals while enzymelevels were approximately 300-fold higher in the spinal meninges of MPSI animals. In repeated experiments, the spinal the spinal cord,rh-iduronidase levels in intrathecally-treated MPS I dogs reached a meanof 160 units/mg or about 13 fold the normal level of 11.7 units/mg(p=0.022, Table 2). Penetration of enzyme was better in the cervical andthoracic regions than in the lumbar spine possibly due to incompletedistribution of enzyme from the cisterna magna injection site. RhIDUlevels in treated MPS I dogs were 17-fold normal in the cervical spinalcord, 18-fold in the thoracic spine, and about 5-fold in the lumbarspine. In the spinal meninges, rh-iduronidase levels reached a mean4,780 units/mg or over 300 fold greater than the normal levels of 15.4units/mg (p=0.0018, Table 2). Even in the animal with the lowest levelof enzyme penetration on average, iduronidase levels in the meningesreached 2,160 units/mg or 140-fold normal levels.

TABLE 2 Iduronidase levels in IT-treated MPS I dogs (~1 mg weekly dose)Untreated/ IT-treated Placebo-treated [range] Normal Ratio IT-treatedCNS Site (n = 4) (n = 5) vs. Normal Brain  277 ± 89.1 11.9 ± 1.95 23.3[203-403] p = 0.0003* Spinal cord Cervical 196 ± 133 11.1 ± 1.69 17.7 [43.3-367.3] Thoracic 224 ± 138 12.0 ± 3.10 18.7 [132.4-428.7] Lumbar59.8 ± 85.9 12.1 ± 2.90 4.9  [8.8-188.0] Average 160 ± 115 11.7 ± 0.5713.7  [73.1-328.0] p = 0.0216* Spinal Meninges Cervical 7030 ± 3480 15.6± 4.85 451   [4060-11,100] Thoracic 5490 ± 4200 14.6 ± 3.34 376[1570-9970] Lumbar 1810 ± 2690 16.1 ± 9.10 112  [95.4-5820] Average  4780 ± 2220.0 15.4 ± 0.76 308 [2160-7580] p = 0.0018* Iduronidaselevels are calculated from mean values for each region for each dogsacrificed. Iduronidase levels are expressed in units of iduronidase permg protein. Means of the means for each animal ± standard deviation areshown. Ranges for each data set are mean values of iduronidase assays ineach tissue type for each dog. *Statistically significant.

Example 5 Intrathecal Treatment of Iduronidase Reduces GAG Levels in MPSI Animals

A significant factor in the debilitation of subjects with lysosomalstorage disorders such as MPS I is the lack of breakdown ofmacromolecules resulting in a build up of glycosaminoglycans in thelysosomes of cells. It is hypothesized that enzyme replacement therapyvia intrathecal injection should enhance the breakdown of GAGs andreturn GAG levels to those comparable to normal individuals.

To test the ability of recombinant iduronidase treatment to ameliorateGAG storage in MPS I subjects, MPS I canines treated as above wereassayed for brain lysosomal levels of glycosaminoglycans. Brain levelsin MPS I animals receiving rh-iduronidase were reduced to normal orbelow normal levels whereas untreated MPS I animals demonstrated GAGlevels approximately 2× that of normal subjects. GAG levels measured inspinal meninges were 7 times normal levels in untreated MPS I animals,but decreased by 57% to 3 times normal levels in MPS I animals receivingintrathecal iduronidase.

GAG levels were also compared in MPS I treated animals receivingintrathecal or IV (either a single bolus, weekly bolus, monthly bolus,quarterly bolus, bolus administered every six months, annual bolus oralternatively, administered continuously) treatment with rh-iduronidase(FIG. 4). GAG levels in MPS I animals receiving IV iduronidase treatmentwere similar to, or slightly higher, than levels observed in untreatedMPS I animals (approximately 10 μg/mg compared to approximately 8 μg/mg,respectively). Intrathecal administration of iduronidase reduced brainGAG levels to below normal, exhibiting approximately 4 μg/mg protein, or2-fold less than untreated MPS I animals.

In further experiments, it was again demonstrated that the many-foldincrease in normal levels of rh-iduronidase activity in the brains oftreated MPS I dogs led to significant decreases in GAG levels relativeto the untreated control MPS I dogs and reached normal GAG levels (Table3). The mean levels of GAG in the brains of MPS I dogs treated withintrathecally with rh-iduronidase were 4.47±0.69 μg/mg dry weightcompared with 8.26±1.23 μg/mg for the untreated MPS I dogs (p=0.0017).The GAG level in the brains of the intrathecally-treated dogs was notsignificantly different from that of untreated normal dogs (5.43±1.95,n=8, p=0.37). The brain GAG levels in intrathecally-treated MPS I dogswas also considerably below that of MPS I dogs treated in prior studieswith IV infusions of rhIDU (10.4±2.14, n=12, Table 3). Since increasingage can result in increased storage, the brain GAG content was alsoplotted against canine age for control, IV-treated and IT-treated dogs.The plot further corroborates the normalization of total GAG forIT-treated dogs when comparing control or IV-treated dogs of comparableage.

Meningeal GAG levels were analyzed in samples derived from the cervical,thoracic and lumbar regions (Table 3). Overall, the mean spinal meningesGAG level of IT-treated dogs decreased 57%, to 15.3 μg/mg (range 9.33 to22.5 μg/mg) compared with an untreated canine mean of 35.9 μg/mg. Thisrepresents a decrease from a level 7-fold normal in untreated MPS I dogsto 3-fold normal in the treated animals and was statisticallysignificant (p=0.009). Samples from the cervical and thoracic meningesoften had better GAG clearance than the more distal lumbar meninges.Mean total GAG levels in the spinal cords of the IT-treated MPS I dogsdecreased to 3.43 μg/mg compared with 5.04 μg/mg for untreated MPS Idogs, but the total levels were relatively low, and the change was notstatistically significant.

TABLE 4 Glycosaminoglycan levels in untreated MPS I, IT-treated MPS I,IV-treated MPS I, and untreated normal dogs MPS I MPS I Ratio IT- MPS IRatio IT- Normal Ratio IT- untreated IT-treated treated to IV-treatedtreated to IV- untreated treated MPS I CNS Site [range] [range]untreated MPS I [range] treated MPS I [range] to normal Brain 8.26 ±1.23 4.47 ± 0.69 0.54 10.4 ± 2.14 0.43 5.43 ± 1.95 0.82 [6.91-9.56][3.63-5.26] p = 0.0017* [7.42-16.6] p = 0.0001* [2.95-8.31] p = 0.37 n =4 n = 4 n = 12 n = 8 Spinal Cord Cervical 3.52 ± 0.56 2.99 ± 0.50 — 1.84± 0.84 [3.12, 3.91] [2.71-3.73] [0.93-2.59] Thoracic 5.50 ± 0.77 2.56 ±0.77 — 2.11 ± 1.03 [4.95, 6.04] [1.90-3.68] [1.29-3.26] Lumbar 6.11 ±1.47 4.75 ± 1.08 — 5.49 ± 0.63 [5.07, 7.15] [3.53-5.73] [4.81-6.06]Average 5.04 ± 0.93 3.43 ± 0.72 0.68 — 3.14 ± 0.81 1.09 [4.38, 5.70][2.72-4.38] p = 0.075 [2.34-3.97] p = 0.64 n = 2 n = 4 n = 3 SpinalMeninges Cervical 20.0 ± 1.98 10.8 ± 2.45 — 4.51 ± 0.51 [18.6, 21.4][9.07-14.4] [3.92-4.86] Thoracic 40.5 ± 0.95 13.4 ± 4.30 — 4.35 ± 1.47[39.8, 41.2] [7.05-16.4] [3.05-5.94] Lumbar 47.2 ± 12.0 21.6 ± 13.8 —5.17 ± 2.53 [38.6, 55.7] [10.6-41.7] [2.33-7.21] Average 35.9 ± 3.0315.3 ± 5.56 0.43 — 4.68 ± 0.97 3.26 [33.8, 38.0]  [9.3-22.5] p = 0.009*[3.75-5.69] p = 0.024* n = 2 n = 4 n = 3 GAG levels are calculated frommean values for each region for each dog sacrificed and are expressed inμg/mg dry weight. Means of the means for each animal ± standarddeviation are shown. IV treatment weekly dose ranged from 0.5 to 2.0mg/kg for 3-15 months. *Statistically significant

These results show that intrathecal treatment of MPS I is an efficientmeans for reducing the debilitating storage in these lysosomal storagedisorders, such as macromolecule build-up in tissue lysosomes, and ismore effective at reducing GAG levels that standard IV administration ofiduronidase enzyme replacement therapy.

Example 6 Reduction of Lysosomal Pathology after Intrathecal rhIDU

In canine MPS I, the most prominent lysosomal storage on brain histologyis present in the perivascular mesenchymal cells that lie close to thebrain capillaries, separated from the bloodstream by the blood-brainbarrier. To determine the extent of the storage disease in MPS Iaffected animals, pathological analysis was performed by electronmicroscopy to detect GAG deposits in brain tissue. MPS I animals weretreated with 1 mg iduronidase in weekly doses (4×) as described above inExample 1.

Tissue taken from untreated MPS I animals with perivascular macrophagedisease demonstrate distinct GAG storage in perithelial cells whereastreated animals exhibit a space around storage vessels, with no GAGstorage (FIG. 5 and FIG. 6). Analysis of neuron disease pathology in MPSI animals reveals that untreated animals show lamellar storage of GAGand gangliosides while iduronidase treated animals exhibit densegranules with minimal storage of the macromolecule (FIG. 7). On electronmicrographs, ultrastructurally the total amount of storage in neurons inuntreated MPS I dogs was modest. The membrane-bound, granular,flocculent, membranous, cytoplasmic and zebra body neuronal storage wasdecreased in the treated MPS I dogs. However, aggregates ofelectron-dense, complex, lipofuscin-like material did remain in thetreated MPS I animals. Brain sections assessed for meningeal disease intreated or untreated MPS I animals demonstrated the presence of largefoam cells in the meninges of untreated animals while meninges ofiduronidase treated animals were free of engorged foam cells containingGAG (FIG. 8). Brain sections of MPS I treated animals did exhibit minorlymphocytic infiltrate into the meninges (FIG. 9). Thus,intrathecally-treated MPS dogs showed a dramatic reduction inperivascular cell storage in both surface and deeper areas of the brain(see FIGS. 5 and 6). GAG storage was also reduced in the glia of thebrains of intrathecally-treated MPS I dogs. Focal reduction inneocortical GAG storage was also seen in three of the four IT-treatedMPS I dogs. GAG storage was also reduced in the spinal meninges oftreated animals on toluidine blue stained thick sections. Spinalmeningeal foam cells were less frequently observed in the four MPS Idogs treated with rhIDU than in the untreated MPS I dogs, and there wassome patchiness to the pattern of clearance.

Overall indications are that intrathecal administration of iduronidasefacilitates clearance of glycosaminoglycans for the brain and meningesof treated subjects, reducing levels back to those observed in normalsubjects. It was also observed that intrathecal delivery of enzymecauses lymphocytic infiltrate into the meninges, perhaps generating animmune response that is effective in clearing inappropriate storage ofmaterials. Analysis of clinical symptoms of lysosomal storage disordersshowed that intrathecal iduronidase treatment of MPS I animals reducedcord compression-induced weakness and resolved nystagmus in theseanimals.

The effectiveness of intrathecal iduronidase treatment over standard IVtechniques indicates that this method of enzyme replacement therapy iseffective for relieving the symptoms of MPS I subjects and is readilyapplicable to other common lysosomal storage disorders described above.

Example 7 Immune Response and Other Adverse Effects

Moderate levels of antibody against rh-iduronidase were detected in boththe serum (up to 202 units/μL) and CSF (up to 82.0 units/μL) of two MPSI dogs and one normal dog treated with rhIDU (Table 4). All three ofthese animals had prior exposure to intravenous rhIDU months beforeentry into the study. For the remaining treated animals, low levels ofantibodies to rhIDU were detected in the serum (3.61 to 40.9 units/μL atstudy end), and lower levels were detected in the CSF (1.39 to 2.28units/μL). There were modest increases in CSF leukocyte counts in thetreated dogs. In all dogs (normal and MPS I) treated with IT rhIDU,there were variable accumulations of B-lymphocytes, plasma cells andother lymphocytes in the meninges of the spinal cord, areas of thespinal dura and around the brain. These dural infiltrates were typicallymost intense around spinal nerve roots and in more severely affectedcases extended into the adjacent extradural fat and connective tissue.In one such case there was also a moderate focal extradural lymphocyticarteritis. There was no meningitis or inflammation in untreated animals,with the exception of one normal dog who received vehicle. Two normaldogs treated with rhIDU developed a mild meningitis. The extent of theCNS inflammatory response varied among dogs and appeared to bedose-related. There were no clinically apparent effects of the immuneresponse observed; the dogs appeared well and active.

TABLE 5 ELISA titer of antibodies to rhIDU in CSF of IT-treated dogs Endof Canine Week 1 Week 2 Week 3 Week 4 treatment IT-treated MPS I dogs Om0.001 0.000 0.166 2.09 2.28 Oz 0.007 0.000 0.046 1.08 1.57 Ta^(†) 0.0000.473 ND 45.0 52.0 Vk^(†) 0.000 4.16 53.1 81.5 82.0 IT-treated normaldogs Xu 0.011 0.008 0.014 1.35 ND Xi 0.012 0.015 0.024 2.49 ND Bu 0.0300.009 0.670 1.43 1.39 Ca^(†) 0.105 3.08 31.1 30.7 32.8 Dv 0.007 0.0000.526 1.57 1.90 Df 0.004 0.000 0.025 0.670 2.25 Titers expressed in ODunits per μL undiluted CSF. ND = not done. *Om, Oz and Bu received 1.08mg, Ta, Vk, and Ca received 1.38 mg, Xu and Xi received 4.14 mg, and Dvand Df received 0.46 mg of IT rhIDU. ^(†)Ta, Vk, and Ca had exposure torhIDU months prior to study entry

The administration of any protein product carries a risk of an immuneresponse, either in the form of chronic antibody formation or aninflammatory response. As seen above, immune responses were observed incanines treated with intrathecal rh-iduronidase. Antibodies toα-L-iduronidase were found in the serum and CSF of three dogs who hadhad exposure to intravenous enzyme prior to entry into this study. Otherthan the lymphoplasmacytic infiltrate, there were no obvious clinicaladverse effects of this immune response.

The intrathecal-based treatments of MPS and other disorders as describedherein may advantageously be administered in combination with a regimenthat produces immune tolerance to the agent being delivered.Particularly contemplated immune tolerance methods include thosedescribed in e.g., U.S. Patent Publication No. 20030211113 and U.S.Patent Publication No. 20040009906, each incorporated herein byreference in its entirety. Further examples of immune toleranceprotocols are provided in Example 9.

Example 8 Treatment of MPS I Subjects with Recombinant Iduronidase

The successful treatment of MPS I canines with recombinant humaniduronidase indicates that intrathecal enzyme replacement therapyprovides effective treatment of human subjects with MPS I.

To treat human MPS I patients with rh-iduronidase, patients withmucopolysaccharidosis I are selected for treatment. The subjects areevaluated at base line and at 6, 10, 14, 18, 22, 26, and at least oncemonthly up to 52 weeks by detailed clinical examinations, magneticresonance imaging of the abdomen and brain, echocardiography,range-of-motion measurements, polysomnography, clinical laboratoryevaluations, measurements of leukocyte α-L-iduronidase activity, andurinary glycosaminoglycan excretion. The subjects should also beassessed for the CNS symptoms that result from lysosomal storagegranules in the brain. Such symptoms include developmental delay and/orregression in development of the subject suffering from the disease,which can be clinically assessed, for example, using Bayley's Scales ofInfant Development II (including monitoring a motor and developmentalquotient), monitoring language or other intellectual and motordevelopments, monitoring evoked potential tests such as auditory orother evoked potential testing. Another symptom, high pressurehydrocephalus caused by the presence of storage granules in the cerebralmeninges near the arachnoid granulations, may be clinically monitoredand assessed using art-recognized methods for determining CSF pressurevia lumbar puncture and/or via an intraventricular catheter. Lysosomalstorage in the cervical meninges near the cord at C1-C5 or elsewherealong the cord also may be clinically assessed, which manifests asprogressive compressive spinal cord compression with lower extremityweakness, loss of bowel and bladder control and sensory deficits alsocould be monitored. Such symptoms may be monitored using e.g.,neurological examination for abnormal Babinski's reflexes, deep tendonreflexes, motor function or sensation. Neurophysiological deficits ofspinal cord compression may be assessed using somatosensory evokedpotentials. Magnetic resonance imaging with or without a contrast agentmay also be used to identify the anatomic location of compression aswell as an evaluation of edema or other indicia of cord injury at thesite of compression. Perivascular storage of lysosomal storage granulescan be assessed by determining the presence of cysts around the vessels,which may also be assessed using MRI scans to determine the size andnumber of such cysts. Monitoring these symptoms before and after thetreatment will allow an assessment of the efficacy of the therapeuticintervention.

Iduronidase is administered to subjects via intrathecal infusion(diluted in normal saline with 0.1 percent human serum albumin) at adose of for example, 1 mg iduronidase per 20 kg of animal weight,delivered weekly. Intrathecal administration is performed via directinjection into the CSF or as in Penn et al., (Neurosurgery. 40:94-9.1997), via a drug pump implanted into the lumbar subarachnoid space,e.g. a Medtronic SYNCHROMED® pump or similar device, for intrathecaldelivery. The pump is implanted according to manufacturer's directionsand may be implanted at any level appropriate for the subject ordisorder being treated. For example, the tip of the pump's catheter maybe placed at the T-10 level in the spine. Subjects are premedicated withdiphenhydramine (0.5 to 1.25 mg per kilogram of body weight).

In initial therapy, iduronidase is given to affected subjects weekly fora four week period. Administration may be continued for extended periodsof time depending on the severity of MPS I disease in the subject beingtreated as well as the age, weight, or sex of the subject. Dosageamounts and duration may be determined by the attending physician.

Subjects are assessed for change in symptoms of MPS I using motor skillstests, MRI analysis of GAG tissue deposits, and GAG levels in urine atthe timepoints noted above. For instance, urinary GAG levels in MPS-Isubjects are compared to normal excretion values. There is a wide rangeof urine GAG values in untreated MPS-I subjects. A greater than 50%reduction in excretion of undegraded GAGs following therapy with therh-iduronidase is a valid means to measure an individual's response totherapy. For example, data is collected measuring the leukocyteiduronidase activity and buccal iduronidase activity before and aftertherapy in MPS I subjects.

Increased motor ability or decreased evidence of GAG deposits in thebrain or GAG levels in urine are indicative that rh-iduronidasetreatment is successfully breaking down excess GAG in the treatedsubjects and relieving symptoms of the disease.

Example 9 Antigen-Specific Tolerance and Intrathecal Enzyme ReplacementTherapy in the Treatment of Lysosomal Storage Disorders

As noted above, intrathecal iduronidase treatment of MPS I affectedanimals resulted in lymphocytic infiltrate into the meninges of treatedanimals. This may be due to an overreaction by the immune system to thepresence of large amounts of foreign antigen delivered to the animal. Toovercome these types of reactions, methods of antigen-specific tolerancehave been used to successfully suppress the immune system. Co-owned,co-pending U.S. patent application No. describes a regimen of treatingMPS I canines which entails induction of antigen-specific tolerance andintravenous administration of iduronidase replacement therapy. Based onthe results described herein, which indicate that intrathecal enzymeadministration is more effective than intravenous injection atdecreasing GAG storage in the brain and relieving clinical symptoms ofMPS I, it follows that use of intrathecal injection coupled with antigenspecific tolerance will provide greater relief to subjects sufferingfrom MPS I.

Subjects with mucopolysaccharidosis I are selected for treatment. Thesubjects are evaluated at base line and at 6, 12, 26, and 52 weeks bydetailed clinical examinations, magnetic resonance imaging of theabdomen and brain, echocardiography, range-of-motion measurements,polysomnography, clinical laboratory evaluations, measurements ofleukocyte α-L-iduronidase activity, and urinary glycosaminoglycanexcretion.

Cyclosporin A (Neoral or Sandimmune) and Azathioprine (Imuran) areobtained from commercial sources. Both drugs are dosed orally at thedose and frequency as follows: CsA Neoral® 12.5 mg/kg/every day dividedbid po; Aza Imuran® 5 mg/kg qod po for two weeks conditioning period.The drugs are then administered at that dose for an additional two weeksin the presence of toleragen. Doses are halved for all drugs each 2weeks after first toleragen infusion. Subjects are monitored for adversereactions, and for CsA peak and trough levels. The CsA is preferably ata level greater than 400 ng/ml.

Recombinant α-L-iduronidase is produced in Chinese-hamster-ovary cellswith the use of bioreactors and standard column chromatography, andextensively analyzed for safety and purity. The activity ofα-L-iduronidase is measured according to the method of Shull et al.supra., or with an assay whose results are reported in SI units (Kakkiset al., Mol Genet Metab. 2001, 72(3):199-208; Kakkis et al., N Engl JMed. 2001; 344(3):182-8). When the latter assay is used, a dose of125,000 U of α-L-iduronidase per kilogram is equivalent to 100 SI unitsper kilogram. Urinary glycosaminoglycan excretion is measured accordingto an adaptation of the method of Bjornsson. Enzyme-linked immunosorbentassays for antibodies to α-L-iduronidase uses a variation of the methodof Shull et al., and Western blotting is performed according to astandard method.

The toleragen is administered by intravenous infusion (diluted in normalsaline with 0.1 percent human serum albumin) at a dose of 0.056 mg/kg,delivered weekly. After tolerization to iduronidase, intrathecaltreatment with the enzyme composition is affected as described herein.In a preferred embodiment, the methods of the present example weretested on dogs treated monthly using a 1 mg injection of rh iduronidaseas a toleragen. After 4 injections over a three month period, the GAGlevels in the brains of these animals were observed as normal. Theadministration protocol is preferably effective such that the 1 mginjections are administered quarterly or every 6 months.

Intrathecal administration for use in the present methods is performedvia direct injection into the CSF or as in Penn et al., (Neurosurgery.40:94-9. 1997), via a drug pump implanted into the lumbar subarachnoidspace, e.g. a Medtronic SYNCHROMED® pump or similar device, forintrathecal delivery. The pump is implanted according to manufacturer'sdirections and may be implanted at any level appropriate for the subjector disorder being treated. For example, the tip of the pump's cathetermay be placed at the T-10 level in the spine. The first dose is givenafter completion of the two week conditioning period, and weeklythereafter. Subjects are premedicated with diphenhydramine (0.5 to 1.25mg per kilogram of body weight).

After induction of tolerance, preferably 12 wee after initiation of theconditioning period, the dose is increased to once weekly, 0.58 mg perkilogram.

Subjects are assessed for change in one or more indicators of thesymptoms of brain disease associated with lysosomal storage diseaseafter treatment with recombinant iduronidase. Such indicators includebut are not limited to changes in development, motor function,maintenance of development over time, decreased CSF pressure, decreasedneurological symptoms by complaint or by examination, decreased cordcompression as examined by MRI analyses of the neck or spine andsomatosensory evoked potentials after treatment with recombinantiduronidase. It is predicted that iduronidase treatment increases thebreakdown of excess GAG in the brain and spinal cord of affectedindividuals and releases the pressure exerted on the spinal cord. Animprovement in motor ability is indicative of a decrease in cordcompression as a result of iduronidase treatment.

Example 10 Intrathecal Treatment of Other Lysosomal Storage Diseases

The above methods are useful in the treatment of human subjectsmanifesting a clinical phenotype of deficiency of any lysosomal enzyme.All subjects manifest some clinical evidence of visceral and soft tissueaccumulation of glycosaminoglycans or other macromolecule with varyingdegrees of functional impairment. The diseases that are treated orprevented using the methods of the present invention are:Mucopolysaccharidosis L1 (MPS II), MPS IIIA, MPS IIIB, MetachromaticLeukodystrophy (MLD), Krabbe, Pompe, Ceroid Lipofuscinosis, Tay-Sachs,Niemann-Pick A and B, Gaucher Disease, and other lysosomal diseases asdescribed above.

For each disease the enzyme administered in the intrathecal enzymereplacement therapy or during the tolerization regimen would comprise aspecific compound or enzyme. For methods involving MPS II, the preferredcompound or enzyme iduronate-2-sulfatase. For methods involving MPS IIA,the preferred compound or enzyme is heparan N-sulfatase. For methodsinvolving MPS IIIB, the preferred compound or enzyme isα-N-acetylglucosaminidase. For methods involving MetachromaticLeukodystropy (MLD), the preferred compound or enzyme is arylsulfataseA. For methods involving Krabbe, the preferred compound or enzyme isgalactosylceramidase. For methods involving Pompe, the preferredcompound or enzyme is acid α-glucosidase. For methods involving CLN, thepreferred compound or enzyme is tripeptidyl peptidase. For methodsinvolving Tay-Sachs, the preferred compound or enzyme is hexosaminidasealpha. For methods involving Niemann-Pick A and B the preferred compoundor enzyme is acid sphingomyelinase.

The enzyme may be administered at doses appropriate for the subjectsbegin treated and are generally delivered based on a mg/kg ratio asdescribed in the Detailed Description. Subjects receiving enzyme aremonitored for enzyme levels in blood and tissue samples and for othersymptoms particular to the lysosomal storage disorder being treated. Forinstance, subjects with Gaucher. Disease (Type 3) who exhibit diminishedmotor skills or myclonic seizures due to aberrant lipid storage aremonitored for improvement in motor skills and decrease in seizurefrequency after intrathecal administration of glucocerebrosidasereplacement therapy.

Improvement in one or more symptoms of a lysosomal storage disorderafter intrathecal administration of an enzyme deficient in the lysosomalstorage disorder demonstrates that this route of administration is a newand useful method for the treatment of lysosomal disorders affectinghuman subjects.

Example 11 Monthly Intrathecal Treatment Regimen

As discussed herein throughout intrathecal administration of rhIDU hasbeen shown to effectively penetrate the CNS. In certain exemplarystudies, weekly doses of approximately 1 mg of rhIDU given intrathecallyhave been shown to penetrate the CNS and reduce glycosaminoglycan (GAG)storage in canine mucopolysaccharidosis I (MPS I). Further studiesdescribed in the present example show that monthly, rather than weeklytreatments, also are effective.

Three MPS I dogs received 4 monthly doses of ˜1 mg IT rhIDU incombination with weekly IV rhIDU. In this combined regimen, it was seenthat iduronidase levels reached 23-fold normal levels in the brain,7-fold in the spinal cord, and 423-fold levels in the meninges of dogstreated monthly, vs. 23-fold, 13-fold, and 300-fold in 4 dogs treatedweekly with intrathecal rhIDU only. Brain GAG reached normal levels inboth regimens. With monthly treatment, a 51% reduction in brain GAGstorage (vs. 46% with weekly IT administration; FIG. 10A) was observed,a 22% reduction in spinal cord GAG (vs. 32% with weekly ITadministration; FIG. 10B), and a 57% reduction in meningeal GAG (vs. 57%with weekly IT administration; FIG. 10C) compared with 4 untreated MPS Idogs. There was no significant difference in iduronidase or GAG levelswith monthly vs. weekly IT rhIDU. As such, monthly intrathecaladministration may be used.

Animals were tested for inflammatory response and for induction oftolerance. One dog developed a lymphoplasmacytic infiltrate in themeninges and a mild antibody response in blood and CSF. One dog hadneurologic signs (see table below) at the start of treatment but thesesigns improved after 4 doses of monthly IT rhIDU with concurrent weeklyIV rhIDU.

BEFORE AFTER Lethargic Alert Ataxic gait No ataxia Gag reflex Gag reflexabsent present Head tilt No head tilt

The second dog had been made tolerant to rhIDU using a novel method(described in e.g., co-owned U.S. application Ser. No. 10/141,668 filedMay 6, 2002 and Ser. No. 10/429,314 filed May 5, 2003, (published asU.S. Patent Publication No. 20030211113 and U.S. Patent Publication No.20040009906, respectively, each incorporated herein by reference in itsentirety) and had little or no detectable immune response in blood andCSF and a very mild meningitis. Treated dogs had diminishedleptomeningeal and perivascular GAG storage histologically. The factthat GAG storage is visibly reduced in perivascular cells, glia, andneocortical leptomeninges in IT treated dogs is depicted in FIG. 11A(untreated showing swollen, foamy, GAG-laden cells) and 11B (treated;thin cells with markedly less GAG storage). With respect to induction oftolerance, the data depicted in FIGS. 12A-12D shows that an animal thathas been preconditioned with an immunosuppressive regimen and becametolerant to rhIDU exhibits a much milder immune response to the rhIDUtherapy. FIGS. 12A and 12C show that in animals treated with rhIDUalone, a lymphocytic and plasmocytic infiltrate develops (FIGS. 12A and12C). Pre-conditioning with a regimen to induce immune tolerance, on theother hand, greatly reduces this response (FIG. 12B and FIG. 12D).

These studies demonstrate that monthly IT rhIDU may be as effective asweekly IT rhIDU in correcting the lysosomal storage in brain andmeninges of canine MPS.

The foregoing describes and exemplifies the invention but is notintended to limit the invention defined by the claims which follow.

I claim:
 1. A method for treating a human suffering from a lysosomalstorage disease, comprising administering directly into cerebrospinalfluid (CSF) of the human a pharmaceutical composition comprising anamount of enzyme effective to ameliorate one or more central nervoussystem (CNS) symptoms of said lysosomal storage disease, wherein theenzyme comprises or has been engineered to comprise a moiety that bindsthe mannose-6-phosphate (M6P) receptor, wherein the moiety is amannose-6-phosphate residue or an IGF-2 polypeptide, wherein the enzymeis human α-N-acetylglucosaminidase and wherein the lysosomal storagedisease is Mucopolysaccharidosis IIIB (MPS IIIB or Sanfilippo SyndromeB), thereby ameliorating one or more CNS symptoms of said lysosomalstorage disease.
 2. The method of claim 1, wherein said administeringcomprises introducing said pharmaceutical composition into a cerebralventricle.
 3. The method of claim 1, wherein said administeringcomprises introducing said pharmaceutical composition into the lumbararea.
 4. The method of claim 1, wherein said administering comprisesintroducing said pharmaceutical composition into the cisterna magna. 5.The method of claim 1, wherein said method further comprisessystemically administering said pharmaceutical composition.
 6. Themethod of claim 5, wherein said systemically administering is achievedby intravenous administration.
 7. The method of claim 1, wherein saidsymptoms are selected from the group consisting of an increased amountof storage granules, an increased amount of glycosaminoglycans,meningeal swelling, spinal cord compression and nystagmus.
 8. The methodof claim 1, wherein the amount is effective to decrease the amount oflysosomal storage granules present in the brain tissue of said human. 9.The method of claim 1, wherein the amount is effective to decrease theamount of lysosomal storage granules present in the meningeal tissue ofsaid human.
 10. The method of claim 1, wherein the enzyme is arecombinant human enzyme.
 11. The method of claim 1, wherein theadministration is once monthly.
 12. The method of claim 1, wherein theadministration is once weekly.
 13. The method of claim 1, wherein saidpharmaceutical composition is intrathecally administered continuallyover a period of at least several days.
 14. The method of claim 1,wherein said pharmaceutical composition is intrathecally administeredcontinually over a period of at least four weeks.
 15. The method ofclaim 1, wherein the moiety is an IGF-2 polypeptide.
 16. The method ofclaim 1, wherein the moiety is a mannose-6-phosphate residue.